EP2926827A2 - Macrocycles peptidomimétiques thérapeutiques - Google Patents

Macrocycles peptidomimétiques thérapeutiques Download PDF

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Publication number
EP2926827A2
EP2926827A2 EP15153712.3A EP15153712A EP2926827A2 EP 2926827 A2 EP2926827 A2 EP 2926827A2 EP 15153712 A EP15153712 A EP 15153712A EP 2926827 A2 EP2926827 A2 EP 2926827A2
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EP
European Patent Office
Prior art keywords
amino acid
peptidomimetic
macrocycle
peptidomimetic macrocycle
cancer
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EP15153712.3A
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German (de)
English (en)
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EP2926827A3 (fr
Inventor
Huw M. Nash
David Allen Annis
Rosana Kapeller-Libermann
Tomi K. Sawyer
Noriyuki Kawahata
Jiawen Han
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Aileron Therapeutics Inc
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Aileron Therapeutics Inc
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Publication of EP2926827A3 publication Critical patent/EP2926827A3/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/64—Cyclic peptides containing only normal peptide links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A61K38/1761—Apoptosis related proteins, e.g. Apoptotic protease-activating factor-1 (APAF-1), Bax, Bax-inhibitory protein(s)(BI; bax-I), Myeloid cell leukemia associated protein (MCL-1), Inhibitor of apoptosis [IAP] or Bcl-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators

Definitions

  • Uncontrolled cell proliferation is implicated in a wide number of disorders ranging from cancer to immunoproliferative diseases. For example, in the U.S. alone, cancer surpasses heart disease as the leading cause of death for the largest fraction of the population ( Journal of the National Cancer Institute, Vol. 97, No. 5, March 2, 2005, p. 330 ) and contributes to more than 500,000 deaths annually. Despite decades of intense research efforts in this area, the treatment of cell proliferative disorders remains a challenge.
  • the present invention addresses this and other needs.
  • the invention provides compositions and methods of treatment based on the surprising finding that certain peptidomimetic macrocycles exhibit unexpected specificity, efficacy and potency when used for treatment of cell proliferative disorders.
  • the present invention provides a method of treating cancer in a human patient in need thereof comprising administering to the patient a peptidomimetic macrocycle, wherein the cancer is selected from the group consisting of small cell lung carcinoma, melanoma, ovarian cancer, prostate cancer, renal cancer, breast cancer, pancreatic cancer, and Ph+ acute lymphocytic leukemia (Ph+ ALL).
  • the peptidomimetic macrocycle comprises an ⁇ -helix.
  • the peptidomimetic macrocycle comprises a BH3 domain.
  • the peptidomimetic macrocycle can be, for example, a BIM polypeptide.
  • an amino acid sequence of the BIM polypeptide is more than about 60% identical to an amino acid sequence IWIAQELR*IGD*FNAYYARR wherein * is a tethered amino acid.
  • the amino acid sequence of the BIM polypeptide is more than about 80% identical to an amino acid sequence IWIAQELR*IGD*FNAYYARR wherein * is a tethered amino acid.
  • an amino acid sequence of said BIM polypeptide may be more than about 95% identical to an amino acid sequence IWIAQELR*IGD*FNAYYARR wherein * is a tethered amino acid.
  • the cancer is at least 2-fold less sensitive to treatment using a corresponding cross-linked BID polypeptide as measured in an in vitro cell viability assay. In other embodiments, the cancer is at least 5-fold less sensitive to treatment using a corresponding cross-linked BID polypeptide as measured in an in vitro cell viability assay. In yet other embodiments, the cancer is at least 8-fold less sensitive to treatment using a corresponding cross-linked BID polypeptide as measured in an in vitro cell viability assay.
  • the cancer is breast cancer, for example an invasive breast carcinoma such as an invasive ductal carcinoma.
  • the cancer is prostate cancer.
  • the cancer is ovarian cancer.
  • the cancer is pancreatic cancer.
  • the cancer is renal cancer.
  • the cancer is Ph+ acute lymphocytic leukemia (Ph+ ALL).
  • the invention also provides a method of treating cancer in a human patient in need thereof comprising administering to the patient a peptidomimetic macrocycle, wherein the cancer is colon cancer.
  • the peptidomimetic macrocycle comprises an ⁇ -helix.
  • the peptidomimetic macrocycle comprises a BH3 domain.
  • the peptidomimetic macrocycle can be, for example, a BID polypeptide.
  • an amino acid sequence of the BID polypeptide is more than about 60% identical to a sequence DIIRNIARHLA*VGD*NleDRSI and wherein * is a tethered amino acid and Nle is norleucine.
  • an amino acid sequence of the BID polypeptide is more than about 80% identical to a sequence DIIRNIARHLA*VGD*NleDRSI wherein * is a tethered amino acid and Nle is norleucine.
  • an amino acid sequence of said BID polypeptide may be more than about 95% identical to a sequence DIIRNIARHLA*VGD*NleDRSI wherein * is a tethered amino acid and Nle is norleucine.
  • the cancer is at least 2-fold less sensitive to treatment using a corresponding cross-linked BIM polypeptide as measured in an in vitro cell viability assay.
  • the cancer is at least 5-fold less sensitive to treatment using a corresponding cross-linked BIM polypeptide as measured in an in vitro cell viability assay. In yet other embodiments, the cancer is at least 8-fold less sensitive to treatment using a corresponding cross-linked BIM polypeptide as measured in an in vitro cell viability assay.
  • a method of treating cancer in a human patient in need thereof comprising administering to the patient a peptidomimetic macrocycle wherein said peptidomimetic macrocycle shows an EC 50 lower than about 5 ⁇ M when tested in an in vitro cell viability assay against a cell line derived from said cancer.
  • the EC 50 may be lower than about 4 ⁇ M.
  • the EC 50 may be lower than about 3 ⁇ M.
  • the EC 50 may be lower than about 2 ⁇ M.
  • the EC 50 may be lower than about 1 ⁇ M.
  • the in vitro assay is performed in the presence of serum.
  • the assay may be performed in 10% human serum.
  • the cancer is selected from the group consisting of ovarian cancer, skin cancer, prostate cancer, renal cancer, breast cancer, pancreatic cancer, small-cell lung cancer, colon cancer, multiple myeloma, Burkitt's lymphoma, acute lymphocytic leukemia (ALL) of T cell lineage or B cell lineage or mixed lineage, chronic lymphocytic leukemia (CLL), cutaneous T cell lymphoma (CTCL), acute myelocytic leukemia (AML), chronic myelocytic leukemia, and follicular lymphoma.
  • ALL acute lymphocytic leukemia
  • CLL chronic lymphocytic leukemia
  • CCL chronic lymphocytic leukemia
  • CCL chronic lymphocytic leukemia
  • AML acute myelocytic leukemia
  • chronic myelocytic leukemia chronic myelocytic leukemia
  • the present invention provides a method of treating cancer in a human patient in need thereof comprising administering to the patient a peptidomimetic macrocycle, wherein the cancer is selected from the group consisting of ovarian cancer, prostate cancer, renal cancer, breast cancer, pancreatic cancer, and Ph+ acute lymphocytic leukemia.
  • the peptidomimetic macrocycle comprises an ⁇ -helix.
  • the peptidomimetic macrocycle comprises a BH3 domain.
  • the peptidomimetic macrocycle can be, for example, a BIM polypeptide.
  • an amino acid sequence of the BIM polypeptide is more than about 60% identical to an amino acid sequence IWIAQELR*IGD*FNAYYARR wherein * is a tethered amino acid.
  • the amino acid sequence of the BIM polypeptide is more than about 80% identical to an amino acid sequence IWIAQELR*IGD*FNAYYARR wherein * is a tethered amino acid.
  • an amino acid sequence of said BIM polypeptide may be more than about 95% identical to an amino acid sequence IWIAQELR*IGD*FNAYYARR wherein * is a tethered amino acid.
  • the cancer is selected from the group consisting of colon cancer, small-cell lung cancer, liver cancer, ovarian cancer, skin cancer, prostate cancer, renal cancer, breast cancer, pancreatic cancer, glioma, multiple myeloma, Burkitt's lymphoma, acute lymphocytic leukemia (ALL) of T cell lineage or B cell lineage or mixed lineage, chronic lymphocytic leukemia (CLL), cutaneous T cell lymphoma (CTCL), acute myelocytic leukemia (AML), chronic myelocytic leukemia and follicular lymphoma.
  • the peptidomimetic macrocycle comprises an ⁇ -helix.
  • the peptidomimetic macrocycle comprises a BH3 domain.
  • the peptidomimetic macrocycle can be, for example, a BID polypeptide.
  • an amino acid sequence of the BID polypeptide is more than about 60% identical to a sequence DIIRNIARHLA*VGD*NleDRSI and wherein * is a tethered amino acid and Nle is norleucine.
  • an amino acid sequence of the BID polypeptide is more than about 80% identical to a sequence DIIRNIARHLA*VGD*NleDRSI wherein * is a tethered amino acid and Nle is norleucine.
  • an amino acid sequence of said BID polypeptide may be more than about 95% identical to a sequence DIIRNIARHLA*VGD*NleDRSI wherein * is a tethered amino acid and Nle is norleucine.
  • the present invention additionally provides a method of treating a disorder in a human patient in need thereof comprising administering to the patient a peptidomimetic macrocycle, comprising a) preparing a peptidomimetic macrocycle by introducing a cross-link between two amino acid residues of a polypeptide; b) testing the peptidomimetic macrocycle for the presence or absence of an immunogenic response; and c) administering the peptidomimetic macrocycle to a patient if said immunogenic response does not cause a substantial side-effect.
  • the non-immunogenicity may be evidenced as minimal antibody response in an in vivo assay in rodents such as mice, in non-human primates, or in humans.
  • a compound which is nonimmunogenic When administered to a human patient, a compound which is nonimmunogenic may induce no substantial or minimal side-effects related to its immunogenicity in the patient.
  • the disorder may be, for example, cancer, a metabolic disease, cardiovascular disease, inflammatory disease or a degenerative disease.
  • the peptidomimetic macrocycle comprises an ⁇ -helix.
  • the peptidomimetic macrocycle comprises a BH3 domain.
  • the peptidomimetic macrocycle can be, for example, a BID polypeptide.
  • an amino acid sequence of the BID polypeptide is more than about 60% identical to a sequence DIIRNIARHLA*VGD*NleDRSI and wherein * is a tethered amino acid and Nle is norleucine.
  • an amino acid sequence of the BID polypeptide is more than about 80% identical to a sequence DIIRNIARHLA*VGD*NleDRSI wherein * is a tethered amino acid and Nle is norleucine.
  • an amino acid sequence of said BID polypeptide may be more than about 95% identical to a sequence DIIRNIARHLA*VGD*NleDRSI wherein * is a tethered amino acid and Nle is norleucine.
  • the peptidomimetic macrocycle may also be, for example, a BIM polypeptide.
  • an amino acid sequence of the BIM polypeptide is more than about 60% identical to an amino acid sequence IWIAQELR*IGD*FNAYYARR wherein * is a tethered amino acid.
  • the amino acid sequence of the BIM polypeptide is more than about 80% identical to an amino acid sequence IWIAQELR*IGD*FNAYYARR wherein * is a tethered amino acid.
  • an amino acid sequence of said BIM polypeptide may be more than about 95% identical to an amino acid sequence IWIAQELR*IGD*FNAYYARR wherein * is a tethered amino acid.
  • the invention provides a method of treating an immunoproliferative disorder in a human patient in need thereof comprising administering to the patient a peptidomimetic macrocycle.
  • the peptidomimetic macrocycle may reduce activated hPBL proliferation by more than about 5%, 10%, 20%, 30%, 40%, or 50% in an in vitro BrdU incorporation assay.
  • the immunoproliferative disease may be, for example, a lymphoproliferative disorder, or an autoimmune disease, for example, systemic lupus erythematosus.
  • the peptidomimetic macrocycle can be, for example, a BID polypeptide.
  • an amino acid sequence of the BID polypeptide is more than about 60% identical to a sequence DIIRNIARHLA*VGD*NleDRSI and wherein * is a tethered amino acid and Nle is norleucine.
  • an amino acid sequence of the BID polypeptide is more than about 80% identical to a sequence DIIRNIARHLA*VGD*NleDRSI wherein * is a tethered amino acid and Nle is norleucine.
  • an amino acid sequence of said BID polypeptide may be more than about 95% identical to a sequence DIIRNIARHLA*VGD*NleDRSI wherein * is a tethered amino acid and Nle is norleucine.
  • the peptidomimetic macrocycle may also be, for example, a BIM polypeptide.
  • an amino acid sequence of the BIM polypeptide is more than about 60% identical to an amino acid sequence IWIAQELR*IGD*FNAYYARR wherein * is a tethered amino acid.
  • the amino acid sequence of the BIM polypeptide is more than about 80% identical to an amino acid sequence IWIAQELR*IGD*FNAYYARR wherein * is a tethered amino acid.
  • an amino acid sequence of said BIM polypeptide may be more than about 95% identical to an amino acid sequence IWIAQELR*IGD*FNAYYARR wherein * is a tethered amino acid.
  • an ⁇ -carbon atom in said peptidomimetic macrocycle may be additionally substituted with independent substituents of formula R-, wherein R- is alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-.
  • R- is alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-.
  • R- is alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-.
  • an ⁇ -carbon atom to which the crosslinker is attached is additionally substituted with a substituent of
  • two ⁇ -carbon atoms in a peptidomimetic macrocycle are additionally substituted with independent substituents of formula R-.
  • two ⁇ -carbon atoms to which the crosslinker is attached are additionally substituted with independent substituents of formula R-.
  • two ⁇ -carbon atoms to which the crosslinker is not attached are additionally substituted with independent substituents of formula R-.
  • R- may be, for example, alkyl such as methyl, ethyl, propyl or isopropyl.
  • the crosslinker may connect two ⁇ -carbon atoms.
  • R- and any portion of the crosslinker taken together form a cyclic structure.
  • the crosslinker is formed of consecutive carbon-carbon bonds. In still other embodiments, the crosslinker contains about 6, 7, 8, 9, 10, 11, 12 or 13 consecutive bonds. In yet other embodiments, the crosslinker comprises at least about 5, 6, 7, 8, or 9 carbon atoms.
  • a method of treating cancer in a human patient in need thereof comprising administering to the patient a peptidomimetic macrocycle wherein said peptidomimetic macrocycle interacts with Mcl-1.
  • the peptidomimetic macrocycle antagonizes the interaction between Mcl-1 and pro-apoptotic proteins such as Bid, Bim, Bax or Bak.
  • the peptidomimetic macrocycles of the invention are used to treat cancer in a human patient wherein the cancer is resistant to ABT-737 or an analog therof, or is resistant to a compound that possesses an affinity greater than 1, 2, 5 or 10 ⁇ M for Mcl-1.
  • the invention further provides a method of treating ABT-737 resistant small cell lung cancer in a human patient in need thereof comprising administering to the patient a peptidomimetic macrocycle, wherein the peptidomimetic macrocycle comprises a BH3 domain.
  • the invention also provides a method of treating prostate cancer in a human patient in need thereof comprising administering to the patient a peptidomimetic macrocycle, wherein the peptidomimetic macrocycle comprises a BH3 domain.
  • the peptidomimetic macrocycle is administered in conjunction with a standard method of care.
  • the standard method of care may, for example, be chemotherapy.
  • the standard method of care may be radiation therapy.
  • the standard method of care is surgery.
  • treating and “to treat” mean to alleviate symptoms, eliminate the causation either on a temporary or permanent basis, or to prevent or slow the appearance of symptoms.
  • treatment includes alleviation, elimination of causation (temporary or permanent) of, or prevention of symptoms and disorders associated with any condition.
  • the treatment may be a pre-treatment as well as a treatment at the onset of symptoms.
  • standard method of care refers to any therapeutic or diagnostic method, compound, or practice which is part of the standard of care for a particular indication.
  • the "standard of care” may be established by any authority such as a health care provider or a national or regional institute for any diagnostic or treatment process that a clinician should follow for a certain type of patient, illness, or clinical circumstance. Exemplary standard of care methods for various type of cancers are provided for instance by the the National Cancer Institute.
  • cell proliferative disorder encompasses cancer, hyperproliferative disorders, neoplastic disorders, immunoproliferative disorders and other disorders.
  • a "cell proliferative disorder” relates to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth.
  • Hyperproliferative and neoplastic disease states may be categorized as pathologic, i.e., characterizing or constituting a disease state, or may be categorized as non-pathologic, i.e., a deviation from normal but not associated with a disease state.
  • metastatic tumor can arise from a multitude of primary tumor types, including but not limited to those of breast, lung, liver, colon and ovarian origin.
  • Primary tumor types including but not limited to those of breast, lung, liver, colon and ovarian origin.
  • Primary tumor types including but not limited to those of breast, lung, liver, colon and ovarian origin.
  • Primary tumor types including but not limited to those of breast, lung, liver, colon and ovarian origin.
  • Primary tumor growth and immunoproliferative diseases include proliferation of cells associated with wound repair.
  • cellular proliferative and/or differentiative disorders include cancer, e.g., carcinoma, sarcoma, or metastatic disorders.
  • the term "derived from” in the context of the relationship between a cell line and a related cancer signifies that the cell line may be established from any cancer in a specific broad category of cancers.
  • microcycle refers to a molecule having a chemical structure including a ring or cycle formed by at least 9 covalently bonded atoms.
  • peptidomimetic macrocycle refers to a compound comprising a plurality of amino acid residues joined by a plurality of peptide bonds and at least one macrocycle-forming linker which forms a macrocycle between a first naturally-occurring or non-naturally-occurring amino acid residue (or analog) and a second naturally-occurring or non-naturally-occurring amino acid residue (or analog) within the same molecule.
  • Peptidomimetic macrocycles include embodiments where the macrocycle-forming linker connects the ⁇ carbon of the first amino acid residue (or analog) to the ⁇ carbon of the second amino acid residue (or analog).
  • the peptidomimetic macrocycles optionally include one or more non-peptide bonds between one or more amino acid residues and/or amino acid analog residues, and optionally include one or more non-naturally-occurring amino acid residues or amino acid analog residues in addition to any which form the macrocycle.
  • the term "stability" refers to the maintenance of a defined secondary structure in solution by a peptidomimetic macrocycle of the invention as measured by circular dichroism, NMR or another biophysical measure, or resistance to proteolytic degradation in vitro or in vivo.
  • Non-limiting examples of secondary structures contemplated in this invention are ⁇ -helices, ⁇ -turns, and ⁇ -pleated sheets.
  • helical stability refers to the maintenance of ⁇ helical structure by a peptidomimetic macrocycle of the invention as measured by circular dichroism or NMR.
  • the peptidomimetic macrocycles of the invention exhibit at least a 1.25, 1.5, 1.75 or 2-fold increase in ⁇ -helicity as determined by circular dichroism compared to a corresponding uncrosslinked polypeptide.
  • ⁇ -amino acid or simply “amino acid” refers to a molecule containing both an amino group and a carboxyl group bound to a carbon which is designated the ⁇ -carbon.
  • Suitable amino acids include, without limitation, both the D-and L-isomers of the naturally-occurring amino acids, as well as non-naturally occurring amino acids prepared by organic synthesis or other metabolic routes. Unless the context specifically indicates otherwise, the term amino acid, as used herein, is intended to include amino acid analogs.
  • naturally occurring amino acid refers to any one of the twenty amino acids commonly found in peptides synthesized in nature, and known by the one letter abbreviations A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y and V.
  • amino acid analog or “non-natural amino acid” refers to a molecule which is structurally similar to an amino acid and which can be substituted for an amino acid in the formation of a peptidomimetic macrocycle.
  • Amino acid analogs include, without limitation, compounds which are structurally identical to an amino acid, as defined herein, except for the inclusion of one or more additional methylene groups between the amino and carboxyl group (e.g., ⁇ -amino ⁇ -carboxy acids), or for the substitution of the amino or carboxy group by a similarly reactive group (e.g., substitution of the primary amine with a secondary or tertiary amine, or substitution or the carboxy group with an ester).
  • non-essential amino acid residue is a residue that can be altered from the wild-type sequence of a polypeptide (e.g., a BH3 domain or the p53 MDM2 binding domain) without abolishing or substantially altering its essential biological or biochemical activity (e.g., receptor binding or activation).
  • An "essential” amino acid residue is a residue that, when altered from the wild-type sequence of the polypeptide, results in abolishing or substantially abolishing the polypeptide's essential biological or biochemical activity.
  • a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., K, R, H), acidic side chains (e.g., D, E), uncharged polar side chains (e.g., G, N, Q, S, T, Y, C), nonpolar side chains (e.g., A, V, L, I, P, F, M, W), beta-branched side chains (e.g., T, V, I) and aromatic side chains (e.g., Y, F, W, H).
  • basic side chains e.g., K, R, H
  • acidic side chains e.g., D, E
  • uncharged polar side chains e.g., G, N, Q, S, T, Y, C
  • nonpolar side chains e.g., A, V, L
  • a predicted nonessential amino acid residue in a BH3 polypeptide is preferably replaced with another amino acid residue from the same side chain family.
  • Other examples of acceptable substitutions are substitutions based on isosteric considerations (e.g. norleucine for methionine) or other properties (e.g. 2-thienylalanine for phenylalanine).
  • member refers to the atoms that form or can form the macrocycle, and excludes substituent or side chain atoms.
  • cyclodecane, 1,2-difluoro-decane and 1,3-dimethyl cyclodecane are all considered ten-membered macrocycles as the hydrogen or fluoro substituents or methyl side chains do not participate in forming the macrocycle.
  • amino acid side chain refers to a moiety attached to the ⁇ -carbon in an amino acid.
  • amino acid side chain for alanine is methyl
  • amino acid side chain for phenylalanine is phenylmethyl
  • amino acid side chain for cysteine is thiomethyl
  • amino acid side chain for aspartate is carboxymethyl
  • amino acid side chain for tyrosine is 4-hydroxyphenylmethyl
  • Other non-naturally occurring amino acid side chains are also included, for example, those that occur in nature ( e.g., an amino acid metabolite) or those that are made synthetically ( e.g. , an ⁇ , ⁇ di-substituted amino acid).
  • ⁇ , ⁇ di-substituted amino acid refers to a molecule or moiety containing both an amino group and a carboxyl group bound to a carbon (the ⁇ -carbon) that is attached to two natural or non-natural amino acid side chains.
  • polypeptide encompasses two or more naturally or non-naturally-occurring amino acids joined by a covalent bond (e.g., an amide bond).
  • Polypeptides as described herein include full length proteins (e.g., fully processed proteins) as well as shorter amino acid sequences (e.g., fragments of naturally-occurring proteins or synthetic polypeptide fragments).
  • microcyclization reagent or "macrocycle-forming reagent” as used herein refers to any reagent which may be used to prepare a peptidomimetic macrocycle of the invention by mediating the reaction between two reactive groups.
  • Reactive groups may be, for example, an azide and alkyne
  • macrocyclization reagents include, without limitation, Cu reagents such as reagents which provide a reactive Cu(I) species, such as CuBr, CuI or CuOTf, as well as Cu(II) salts such as Cu(CO 2 CH 3 ) 2 , CuSO 4 , and CuCl 2 that can be converted in situ to an active Cu(I) reagent by the addition of a reducing agent such as ascorbic acid or sodium ascorbate.
  • Macrocyclization reagents may additionally include, for example, Ru reagents known in the art such as Cp*RuCl(PPh 3 ) 2 , [Cp*RuCl] 4 or other Ru reagents which may provide a reactive Ru(II) species.
  • the reactive groups are terminal olefins.
  • the macrocyclization reagents or macrocycle-forming reagents are metathesis catalysts including, but not limited to, stabilized, late transition metal carbene complex catalysts such as Group VIII transition metal carbene catalysts.
  • such catalysts are Ru and Os metal centers having a +2 oxidation state, an electron count of 16 and pentacoordinated.
  • the reactive groups are thiol groups.
  • the macrocyclization reagent is, for example, a linker functionalized with two thiol-reactive groups such as halogen groups.
  • halo or halogen refers to fluorine, chlorine, bromine or iodine or a radical thereof.
  • alkyl refers to a hydrocarbon chain that is a straight chain or branched chain, containing the indicated number of carbon atoms. For example, C 1 -C 10 indicates that the group has from 1 to 10 (inclusive) carbon atoms in it. In the absence of any numerical designation, “alkyl” is a chain (straight or branched) having 1 to 20 (inclusive) carbon atoms in it.
  • alkylene refers to a divalent alkyl (i.e., -R-).
  • alkenyl refers to a hydrocarbon chain that is a straight chain or branched chain having one or more carbon-carbon double bonds.
  • the alkenyl moiety contains the indicated number of carbon atoms. For example, C 2 -C 10 indicates that the group has from 2 to 10 (inclusive) carbon atoms in it.
  • lower alkenyl refers to a C 2 -C 6 alkenyl chain. In the absence of any numerical designation, "alkenyl” is a chain (straight or branched) having 2 to 20 (inclusive) carbon atoms in it.
  • alkynyl refers to a hydrocarbon chain that is a straight chain or branched chain having one or more carbon-carbon triple bonds.
  • the alkynyl moiety contains the indicated number of carbon atoms.
  • C 2 -C 10 indicates that the group has from 2 to 10 (inclusive) carbon atoms in it.
  • lower alkynyl refers to a C 2 -C 6 alkynyl chain.
  • alkynyl is a chain (straight or branched) having 2 to 20 (inclusive) carbon atoms in it.
  • aryl refers to a 6-carbon monocyclic or 10-carbon bicyclic aromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring are substituted by a substituent. Examples of aryl groups include phenyl, naphthyl and the like.
  • arylalkyl or the term “aralkyl” refers to alkyl substituted with an aryl.
  • arylalkoxy refers to an alkoxy substituted with aryl.
  • Arylalkyl refers to an aryl group, as defined above, wherein one of the aryl group's hydrogen atoms has been replaced with a C 1 -C 5 alkyl group, as defined above.
  • Representative examples of an arylalkyl group include, but are not limited to, 2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 2-ethylphenyl, 3-ethylphenyl, 4-ethylphenyl, 2-propylphenyl, 3-propylphenyl, 4-propylphenyl, 2-butylphenyl, 3-butylphenyl, 4-butylphenyl, 2-pentylphenyl, 3-pentylphenyl, 4-pentylphenyl, 2-isopropylphenyl, 3-isopropylphenyl, 4-isopropylphenyl, 2-isobutylphenyl, 3-isobutylphenyl, 4-isopropylphenyl
  • Arylamido refers to an aryl group, as defined above, wherein one of the aryl group's hydrogen atoms has been replaced with one or more -C(O)NH 2 groups.
  • Representative examples of an arylamido group include 2-C(O)NH2-phenyl, 3-C(O)NH 2 -phenyl, 4-C(O)NH 2 -phenyl, 2-C(O)NH 2 -pyridyl, 3-C(O)NH 2 -pyridyl, and 4-C(O)NH 2 -pyridyl,
  • Alkylheterocycle refers to a C 1 -C 5 alkyl group, as defined above, wherein one of the C 1 -C 5 alkyl group's hydrogen atoms has been replaced with a heterocycle.
  • Representative examples of an alkylheterocycle group include, but are not limited to, -CH 2 CH 2 -morpholine, -CH 2 CH 2 -piperidine, -CH 2 CH 2 CH 2 -morpholine, and -CH 2 CH 2 CH 2 -imidazole.
  • Alkylamido refers to a C 1 -C 5 alkyl group, as defined above, wherein one of the C 1 -C 5 alkyl group's hydrogen atoms has been replaced with a -C(O)NH 2 group.
  • Alkanol refers to a C 1 -C 5 alkyl group, as defined above, wherein one of the C 1 -C 5 alkyl group's hydrogen atoms has been replaced with a hydroxyl group.
  • Representative examples of an alkanol group include, but are not limited to, -CH 2 OH, -CH 2 CH 2 OH, -CH 2 CH 2 CH 2 OH, -CH 2 CH 2 CH 2 CH 2 OH, -CH 2 CH 2 CH 2 CH 2 CH 2 OH, -CH 2 CH(OH)CH 3 , -CH 2 CH(OH)CH 2 CH 3 , -CH(OH)CH 3 and -C(CH 3 ) 2 CH 2 OH.
  • Alkylcarboxy refers to a C 1 -C 5 alkyl group, as defined above, wherein one of the C 1 -C 5 alkyl group's hydrogen atoms has been replaced with a --COOH group.
  • Representative examples of an alkylcarboxy group include, but are not limited to, -CH 2 COOH, -CH 2 CH 2 COOH, -CH 2 CH 2 CH 2 COOH, - CH 2 CH 2 CH 2 COOH, -CH 2 CH(COOH)CH 3 , -CH 2 CH 2 CH 2 CH 2 COOH, -CH 2 CH(COOH)CH 2 CH 3 , - CH(COOH)CH 2 CH 3 and -C(CH 3 ) 2 CH 2 COOH.
  • cycloalkyl as employed herein includes saturated and partially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons, preferably 3 to 8 carbons, and more preferably 3 to 6 carbons, wherein the cycloalkyl group additionally is optionally substituted.
  • Some cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.
  • heteroaryl refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of O, N, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2, 3, or 4 atoms of each ring are substituted by a substituent.
  • heteroaryl groups include pyridyl, furyl or furanyl, imidazolyl, benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, quinolinyl, indolyl, thiazolyl, and the like.
  • heteroarylalkyl or the term “heteroaralkyl” refers to an alkyl substituted with a heteroaryl.
  • heteroarylalkoxy refers to an alkoxy substituted with heteroaryl.
  • heteroarylalkyl or the term “heteroaralkyl” refers to an alkyl substituted with a heteroaryl.
  • heteroarylalkoxy refers to an alkoxy substituted with heteroaryl.
  • heterocyclyl refers to a nonaromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of O, N, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2 or 3 atoms of each ring are substituted by a substituent.
  • heterocyclyl groups include piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, and the like.
  • substituted refers to a group replacing a second atom or group such as a hydrogen atom on any molecule, compound or moiety.
  • Suitable substituents include, without limitation, halo, hydroxy, mercapto, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, thioalkoxy, aryloxy, amino, alkoxycarbonyl, amido, carboxy, alkanesulfonyl, alkylcarbonyl, and cyano groups.
  • the compounds of this invention contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of these compounds are included in the present invention unless expressly provided otherwise.
  • the compounds of this invention are also represented in multiple tautomeric forms, in such instances, the invention includes all tautomeric forms of the compounds described herein (e.g., if alkylation of a ring system results in alkylation at multiple sites, the invention includes all such reaction products). All such isomeric forms of such compounds are included in the present invention unless expressly provided otherwise. All crystal forms of the compounds described herein are included in the present invention unless expressly provided otherwise.
  • the terms “increase” and “decrease” mean, respectively, to cause a statistically significantly (i.e., p ⁇ 0.1) increase or decrease of at least 5%.
  • variable is equal to any of the values within that range.
  • variable is equal to any integer value within the numerical range, including the end-points of the range.
  • variable is equal to any real value within the numerical range, including the end-points of the range.
  • a variable which is described as having values between 0 and 2 takes the values 0, 1 or 2 if the variable is inherently discrete, and takes the values 0.0, 0.1, 0.01, 0.001, or any other real values ⁇ 0 and ⁇ 2 if the variable is inherently continuous.
  • on average represents the mean value derived from performing at least three independent replicates for each data point.
  • biological activity encompasses structural and functional properties of a macrocycle of the invention.
  • Biological activity is, for example, structural stability, alpha-helicity, affinity for a target, resistance to proteolytic degradation, cell penetrability, intracellular stability, in vivo stability, or any combination thereof.
  • any protein or polypeptide with a known primary amino acid sequence which contains a helical structure believed to impart biological activity is the subject of the present invention.
  • the sequence of the polypeptide can be analyzed and amino acid analogs containing groups reactive with macrocyclization reagents can be substituted at the appropriate positions.
  • the appropriate positions are determined by ascertaining which molecular surface(s) of the secondary structure is (are) required for biological activity and, therefore, across which other surface(s) the macrocycle forming linkers of the invention can form a macrocycle without sterically blocking the surface(s) required for biological activity.
  • Such determinations are made using methods such as X-ray crystallography of complexes between the secondary structure and a natural binding partner to visualize residues (and surfaces) critical for activity; by sequential mutagenesis of residues in the secondary structure to functionally identify residues (and surfaces) critical for activity; or by other methods.
  • the appropriate amino acids are substituted with the amino acids analogs and macrocycle-forming linkers of the invention.
  • one surface of the helix e.g., a molecular surface extending longitudinally along the axis of the helix and radially 45-135° about the axis of the helix
  • a macrocycle-forming linker is designed to link two ⁇ -carbons of the helix while extending longitudinally along the surface of the helix in the portion of that surface not directly required for activity.
  • the peptide sequence is derived from the BCL-2 family of proteins.
  • the BCL-2 family is defined by the presence of up to four conserved BCL-2 homology (BH) domains designated BH1, BH2, BH3, and BH4, all of which include ⁇ -helical segments ( Chittenden et al. (1995), EMBO 14:5589 ; Wang et al. (1996), Genes Dev. 10:2859 ).
  • Anti-apoptotic proteins, such as BCL-2 and BCL-X L display sequence conservation in all BH domains.
  • Pro-apoptotic proteins are divided into "multidomain” family members (e.g., BAK, BAX), which possess homology in the BH1, BH2, and BH3 domains, and "BH3-domain only” family members (e.g., BID, BAD, BIM, BIK, NOXA, PUMA), that contain sequence homology exclusively in the BH3 amphipathic ⁇ -helical segment.
  • BCL-2 family members have the capacity to form homo- and heterodimers, suggesting that competitive binding and the ratio between pro- and anti-apoptotic protein levels dictates susceptibility to death stimuli.
  • Anti-apoptotic proteins function to protect cells from pro-apoptotic excess, i.e., excessive programmed cell death.
  • Additional "security” measures include regulating transcription of pro-apoptotic proteins and maintaining them as inactive conformers, requiring either proteolytic activation, dephosphorylation, or ligand-induced conformational change to activate pro-death functions.
  • death signals received at the plasma membrane trigger apoptosis via a mitochondrial pathway.
  • the mitochondria can serve as a gatekeeper of cell death by sequestering cytochrome c, a critical component of a cytosolic complex which activates caspase 9, leading to fatal downstream proteolytic events.
  • Multidomain proteins such as BCL-2/BCL-X L and BAK/BAX play dueling roles of guardian and executioner at the mitochondrial membrane, with their activities further regulated by upstream BH3-only members of the BCL-2 family.
  • BID is a member of the BH3-domain only family of pro-apoptotic proteins, and transmits death signals received at the plasma membrane to effector pro-apoptotic proteins at the mitochondrial membrane.
  • BID has the capability of interacting with both pro- and anti-apoptotic proteins, and upon activation by caspase 8, triggers cytochrome c release and mitochondrial apoptosis.
  • amphipathic ⁇ -helical BH3 segment of pro-apoptotic family members may function as a death domain and thus may represent a critical structural motif for interacting with multidomain apoptotic proteins.
  • Structural studies have shown that the BH3 helix can interact with anti-apoptotic proteins by inserting into a hydrophobic groove formed by the interface of BH1, 2 and 3 domains.
  • Activated BID can be bound and sequestered by anti-apoptotic proteins (e.g., BCL-2 and BCL-X L ) and can trigger activation of the pro-apoptotic proteins BAX and BAK, leading to cytochrome c release and a mitochondrial apoptosis program.
  • BAD is also a BH3-domain only pro-apoptotic family member whose expression triggers the activation of BAX/BAK.
  • BAD displays preferential binding to anti-apoptotic family members, BCL-2 and BCL-X L .
  • BAD BH3 domain exhibits high affinity binding to BCL-2
  • BAD BH3 peptide is unable to activate cytochrome c release from mitochondria in vitro, suggesting that BAD is not a direct activator of BAX/BAK.
  • Mitochondria that over-express BCL-2 are resistant to BID-induced cytochrome c release, but co-treatment with BAD can restore BID sensitivity.
  • Induction of mitochondrial apoptosis by BAD appears to result from either: (1) displacement of BAX/BAK activators, such as BID and BID-like proteins, from the BCL-2/BCL-XL binding pocket, or (2) selective occupation of the BCL-2/BCL-XL binding pocket by BAD to prevent sequestration of BID-like proteins by anti-apoptotic proteins.
  • BID and BID-like proteins are two classes of BH3-domain only proteins.
  • a polypeptide of the invention contains one crosslink. In other embodiments of the method, said polypeptide contains two cross-links. In some embodiments of the method, one crosslink connects two ⁇ -carbon atoms. In other embodiments of the method, one ⁇ -carbon atom to which one crosslink is attached is substituted with a substituent of formula R-. In another embodiment of the method, two ⁇ -carbon atoms to which one crosslink is attached are substituted with independent substituents of formula R-. In one embodiment of the methods of the invention, R- is alkyl. For example, R- is methyl. Alternatively, R- and any portion of one crosslink taken together can form a cyclic structure. In another embodiment of the method, one crosslink is formed of consecutive carbon-carbon bonds. For example, one crosslink may comprise at least 8, 9, 10, 11, or 12 consecutive bonds. In other embodiments, one crosslink may comprise at least 7, 8, 9, 10, or 11 carbon atoms.
  • the crosslinked polypeptide comprises an ⁇ -helical domain of a BCL-2 family member.
  • the crosslinked polypeptide comprises a BH3 domain.
  • the crosslinked polypeptide comprises at least 60%, 70%, 80%, 85%, 90% or 95% of any of the sequences in Tables 1, 2, 3 and 4.
  • the crosslinked polypeptide penetrates cell membranes by an energy-dependent process and binds to an intracellular target.
  • said helical polypeptide contains one crosslink. In other embodiments, said helical polypeptide contains two cross-links.
  • one crosslink connects two ⁇ -carbon atoms.
  • one ⁇ -carbon atom to which one crosslink is attached is substituted with a substituent of formula R-.
  • two ⁇ -carbon atoms to which one crosslink is attached are substituted with independent substituents of formula R-.
  • R- is alkyl.
  • R- is methyl.
  • R- and any portion of one crosslink taken together can form a cyclic structure.
  • one crosslink is formed of consecutive carbon-carbon bonds.
  • one crosslink may comprise at least 8, 9, 10, 11, or 12 consecutive bonds.
  • one crosslink may comprise at least 7, 8, 9, 10, or 11 carbon atoms.
  • the crosslinked polypeptide comprises an ⁇ -helical domain of a BCL-2 family member.
  • the crosslinked polypeptide comprises a BH3 domain.
  • the crosslinked polypeptide comprises at least 60%, 70%, 80%, 85%, 90% or 95% of any of the sequences in Tables 1, 2, 3 and 4.
  • the crosslinked polypeptide penetrates cell membranes by an energy-dependent process and binds to an intracellular target.
  • the peptidomimetic macrocycles of the invention have the Formula (I): wherein:
  • At least one of R 1 and R 2 is alkyl, unsubstituted or substituted with halo-. In another example, both R 1 and R 2 are independently alkyl, unsubstituted or substituted with halo-. In some embodiments, at least one of R 1 and R 2 is methyl. In other embodiments, R 1 and R 2 are methyl.
  • x+y+z is at least 3. In other embodiments of the invention, x+y+z is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • Each occurrence of A, B, C, D or E in a macrocycle or macrocycle precursor of the invention is independently selected.
  • a sequence represented by the formula [A] x when x is 3, encompasses embodiments where the amino acids are not identical, e.g. Gln-Asp-Ala as well as embodiments where the amino acids are identical, e.g. Gln-Gln-Gln. This applies for any value of x, y, or z in the indicated ranges.
  • the peptidomimetic macrocycle of the invention comprises a secondary structure which is an ⁇ -helix and R 8 is -H, allowing intrahelical hydrogen bonding.
  • at least one of A, B, C, D or E is an ⁇ , ⁇ -disubstituted amino acid.
  • B is an ⁇ , ⁇ -disubstituted amino acid.
  • at least one of A, B, C, D or E is 2-aminoisobutyric acid.
  • at least one of A, B, C, D or E is
  • the length of the macrocycle-forming linker L as measured from a first C ⁇ to a second C ⁇ is selected to stabilize a desired secondary peptide structure, such as an ⁇ -helix formed by residues of the peptidomimetic macrocycle including, but not necessarily limited to, those between the first C ⁇ to a second C ⁇ .
  • the peptidomimetic macrocycle of Formula (I) is:
  • each R 1 and R 2 is independently independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-.
  • the peptidomimetic macrocycle of Formula (I) is: or
  • the peptidomimetic macrocycle of Formula (I) is a compound of any of the formulas shown below: wherein "AA” represents any natural or non-natural amino acid side chain and " " is [D] v , [E] w as defined above, and n is an integer between 0 and 20, 50, 100, 200, 300, 400 or 500. In some embodiments, n is 0. In other embodiments, n is less than 50.
  • peptidomimetic macrocycles of the invention include analogs of the macrocycles shown above.
  • the peptidomimetic macrocycles of the invention have the Formula (II): wherein:
  • At least one of R 1 and R 2 is alkyl, unsubstituted or substituted with halo-. In another example, both R 1 and R 2 are independently alkyl, unsubstituted or substituted with halo-. In some embodiments, at least one of R 1 and R 2 is methyl. In other embodiments, R 1 and R 2 are methyl.
  • x+y+z is at least 3. In other embodiments of the invention, x+y+z is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • Each occurrence of A, B, C, D or E in a macrocycle or macrocycle precursor of the invention is independently selected.
  • a sequence represented by the formula [A] x when x is 3, encompasses embodiments where the amino acids are not identical, e.g. Gln-Asp-Ala as well as embodiments where the amino acids are identical, e.g. Gln-Gln-Gln. This applies for any value of x, y, or z in the indicated ranges.
  • the peptidomimetic macrocycle of the invention comprises a secondary structure which is an ⁇ -helix and R 8 is -H, allowing intrahelical hydrogen bonding.
  • at least one of A, B, C, D or E is an ⁇ , ⁇ -disubstituted amino acid.
  • B is an ⁇ , ⁇ -disubstituted amino acid.
  • at least one of A, B, C, D or E is 2-aminoisobutyric acid.
  • at least one of A, B, C, D or E is
  • the length of the macrocycle-forming linker L as measured from a first C ⁇ to a second C ⁇ is selected to stabilize a desired secondary peptide structure, such as an ⁇ -helix formed by residues of the peptidomimetic macrocycle including, but not necessarily limited to, those between the first C ⁇ to a second C ⁇ .
  • the invention provides peptidomimetic macrocycles of Formula (III): wherein:
  • At least one of R 1 and R 2 is alkyl, unsubstituted or substituted with halo-. In another example, both R 1 and R 2 are independently alkyl, unsubstituted or substituted with halo-. In some embodiments, at least one of R 1 and R 2 is methyl. In other embodiments, R 1 and R 2 are methyl.
  • x+y+z is at least 3. In other embodiments of the invention, x+y+z is 3, 4, 5, 6, 7, 8, 9 or 10.
  • Each occurrence of A, B, C, D or E in a macrocycle or macrocycle precursor of the invention is independently selected.
  • a sequence represented by the formula [A] x when x is 3, encompasses embodiments where the amino acids are not identical, e.g. Gln-Asp-Ala as well as embodiments where the amino acids are identical, e.g. Gln-Gln-Gln. This applies for any value of x, y, or z in the indicated ranges.
  • the peptidomimetic macrocycle of the invention comprises a secondary structure which is an ⁇ -helix and R 8 is -H, allowing intrahelical hydrogen bonding.
  • at least one of A, B, C, D or E is an ⁇ , ⁇ -disubstituted amino acid.
  • B is an ⁇ , ⁇ -disubstituted amino acid.
  • at least one of A, B, C, D or E is 2-aminoisobutyric acid.
  • at least one of A, B, C, D or E is
  • the length of the macrocycle-forming linker [-L 1 -S-L 2 -S-L 3 -] as measured from a first C ⁇ to a second C ⁇ is selected to stabilize a desired secondary peptide structure, such as an ⁇ -helix formed by residues of the peptidomimetic macrocycle including, but not necessarily limited to, those between the first C ⁇ to a second C ⁇ .
  • Macrocycles or macrocycle precursors are synthesized, for example, by solution phase or solid-phase methods, and can contain both naturally-occurring and non-naturally-occurring amino acids. See, for example, Hunt, "The Non-Protein Amino Acids” in Chemistry and Biochemistry of the Amino Acids, edited by G.C. Barrett, Chapman and Hall, 1985 .
  • the thiol moieties are the side chains of the amino acid residues L-cysteine, D-cysteine, ⁇ -methyl-L cysteine, ⁇ -methyl-D-cysteine, L-homocysteine, D-homocysteine, ⁇ -methyl-L-homocysteine or ⁇ -methyl-D-homocysteine.
  • a bis-alkylating reagent is of the general formula X-L 2 -Y wherein L 2 is a linker moiety and X and Y are leaving groups that are displaced by -SH moieties to form bonds with L 2 .
  • X and Y are halogens such as I, Br, or Cl.
  • D and/or E in the compound of Formula I, II or III are further modified in order to facilitate cellular uptake.
  • lipidating or PEGylating a peptidomimetic macrocycle facilitates cellular uptake, increases bioavailability, increases blood circulation, alters pharmacokinetics, decreases immunogenicity and/or decreases the needed frequency of administration.
  • At least one of [D] and [E] in the compound of Formula I, II or III represents a moiety comprising an additional macrocycle-forming linker such that the peptidomimetic macrocycle comprises at least two macrocycle-forming linkers.
  • a peptidomimetic macrocycle comprises two macrocycle-forming linkers.
  • any of the macrocycle-forming linkers described herein may be used in any combination with any of the sequences shown in Tables 1-4 and also with any of the R- substituents indicated herein.
  • the peptidomimetic macrocycle comprises at least one ⁇ -helix motif.
  • A, B and/or C in the compound of Formula I, II or III include one or more ⁇ -helices.
  • ⁇ -helices include between 3 and 4 amino acid residues per turn.
  • the ⁇ -helix of the peptidomimetic macrocycle includes 1 to 5 turns and, therefore, 3 to 20 amino acid residues.
  • the ⁇ -helix includes 1 turn, 2 turns, 3 turns, 4 turns, or 5 turns.
  • the macrocycle-forming linker stabilizes an ⁇ -helix motif included within the peptidomimetic macrocycle.
  • the length of the macrocycle-forming linker L from a first C ⁇ to a second C ⁇ is selected to increase the stability of an ⁇ -helix.
  • the macrocycle-forming linker spans from 1 turn to 5 turns of the ⁇ -helix. In some embodiments, the macrocycle-forming linker spans approximately 1 turn, 2 turns, 3 turns, 4 turns, or 5 turns of the ⁇ -helix. In some embodiments, the length of the macrocycle-forming linker is approximately 5 ⁇ to 9 ⁇ per turn of the ⁇ -helix, or approximately 6 ⁇ to 8 ⁇ per turn of the ⁇ -helix.
  • the length is equal to approximately 5 carbon-carbon bonds to 13 carbon-carbon bonds, approximately 7 carbon-carbon bonds to 11 carbon-carbon bonds, or approximately 9 carbon-carbon bonds.
  • the length is equal to approximately 8 carbon-carbon bonds to 16 carbon-carbon bonds, approximately 10 carbon-carbon bonds to 14 carbon-carbon bonds, or approximately 12 carbon-carbon bonds.
  • the macrocycle-forming linker spans approximately 3 turns of an ⁇ -helix, the length is equal to approximately 14 carbon-carbon bonds to 22 carbon-carbon bonds, approximately 16 carbon-carbon bonds to 20 carbon-carbon bonds, or approximately 18 carbon-carbon bonds.
  • the length is equal to approximately 20 carbon-carbon bonds to 28 carbon-carbon bonds, approximately 22 carbon-carbon bonds to 26 carbon-carbon bonds, or approximately 24 carbon-carbon bonds.
  • the macrocycle-forming linker spans approximately 5 turns of an ⁇ -helix, the length is equal to approximately 26 carbon-carbon bonds to 34 carbon-carbon bonds, approximately 28 carbon-carbon bonds to 32 carbon-carbon bonds, or approximately 30 carbon-carbon bonds.
  • the linkage contains approximately 4 atoms to 12 atoms, approximately 6 atoms to 10 atoms, or approximately 8 atoms.
  • the linkage contains approximately 7 atoms to 15 atoms, approximately 9 atoms to 13 atoms, or approximately 11 atoms.
  • the linkage contains approximately 13 atoms to 21 atoms, approximately 15 atoms to 19 atoms, or approximately 17 atoms.
  • the linkage contains approximately 19 atoms to 27 atoms, approximately 21 atoms to 25 atoms, or approximately 23 atoms.
  • the linkage contains approximately 25 atoms to 33 atoms, approximately 27 atoms to 31 atoms, or approximately 29 atoms.
  • the resulting macrocycle forms a ring containing approximately 17 members to 25 members, approximately 19 members to 23 members, or approximately 21 members.
  • the macrocycle-forming linker spans approximately 2 turns of the ⁇ -helix, the resulting macrocycle forms a ring containing approximately 29 members to 37 members, approximately 31 members to 35 members, or approximately 33 members.
  • the resulting macrocycle forms a ring containing approximately 44 members to 52 members, approximately 46 members to 50 members, or approximately 48 members.
  • the resulting macrocycle forms a ring containing approximately 59 members to 67 members, approximately 61 members to 65 members, or approximately 63 members.
  • the macrocycle-forming linker spans approximately 5 turns of the ⁇ -helix, the resulting macrocycle forms a ring containing approximately 74 members to 82 members, approximately 76 members to 80 members, or approximately 78 members.
  • the invention provides peptidomimetic macrocycles of Formula (IV) or (IVa): wherein:
  • At least one of R 1 and R 2 is alkyl, unsubstituted or substituted with halo-. In another example, both R 1 and R 2 are independently alkyl, unsubstituted or substituted with halo-. In some embodiments, at least one of R 1 and R 2 is methyl. In other embodiments, R 1 and R 2 are methyl.
  • x+y+z is at least 3. In other embodiments of the invention, x+y+z is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • Each occurrence of A, B, C, D or E in a macrocycle or macrocycle precursor of the invention is independently selected.
  • a sequence represented by the formula [A] x when x is 3, encompasses embodiments where the amino acids are not identical, e.g. Gln-Asp-Ala as well as embodiments where the amino acids are identical, e.g. Gln-Gln-Gln. This applies for any value of x, y, or z in the indicated ranges.
  • the peptidomimetic macrocycle of the invention comprises a secondary structure which is an ⁇ -helix and R 8 is -H, allowing intrahelical hydrogen bonding.
  • at least one of A, B, C, D or E is an ⁇ , ⁇ -disubstituted amino acid.
  • B is an ⁇ , ⁇ -disubstituted amino acid.
  • at least one of A, B, C, D or E is 2-aminoisobutyric acid.
  • at least one of A, B, C, D or E is
  • the length of the macrocycle-forming linker L as measured from a first C ⁇ to a second C ⁇ is selected to stabilize a desired secondary peptide structure, such as an ⁇ -helix formed by residues of the peptidomimetic macrocycle including, but not necessarily limited to, those between the first C ⁇ to a second C ⁇ .
  • Peptidomimetic macrocycles of the invention may be prepared by any of a variety of methods known in the art.
  • any of the residues indicated by "X" in Tables 1, 2, 3 or 4 may be substituted with a residue capable of forming a crosslinker with a second residue in the same molecule or a precursor of such a residue.
  • the peptidomimetic macrocyles of the invention are of Formula IV or IVa. Methods for the preparation of such macrocycles are described, for example, in US Patent No. 7,202,332 .
  • the synthesis of these peptidomimetic macrocycles involves a multi-step process that features the synthesis of a peptidomimetic precursor containing an azide moiety and an alkyne moiety; followed by contacting the peptidomimetic precursor with a macrocyclization reagent to generate a triazole-linked peptidomimetic macrocycle.
  • Macrocycles or macrocycle precursors are synthesized, for example, by solution phase or solid-phase methods, and can contain both naturally-occurring and non-naturally-occurring amino acids. See, for example, Hunt, "The Non-Protein Amino Acids" in Chemistry and Biochemistry of the Amino Acids, edited by G.C. Barrett, Chapman and Hall, 1985 .
  • an azide is linked to the ⁇ -carbon of a residue and an alkyne is attached to the ⁇ -carbon of another residue.
  • the azide moieties are azido-analogs of amino acids L-lysine, D-lysine, alpha-methyl-L-lysine, alpha-methyl-D-lysine, L-ornithine, D-ornithine, alpha-methyl-L-ornithine or alpha-methyl-D-ornithine.
  • the alkyne moiety is L-propargylglycine.
  • the alkyne moiety is an amino acid selected from the group consisting of L-propargylglycine, D-propargylglycine, (S)-2-amino-2-methyl-4-pentynoic acid, (R)-2-amino-2-methyl-4-pentynoic acid, (S)-2-amino-2-methyl-5-hexynoic acid, (R)-2-amino-2-methyl-5-hexynoic acid, (S)-2-amino-2-methyl-6-heptynoic acid, (R)-2-amino-2-methyl-6-heptynoic acid, (S)-2-amino-2-methyl-7-octynoic acid, (R)-2-amino-2-methyl-7-octynoic acid, (S)-2-amino-2-methyl-8-nonynoic acid and (R)-2-amino-2-methyl-8-nonynoic acid.
  • the invention provides a method for synthesizing a peptidomimetic macrocycle, the method comprising the steps of contacting a peptidomimetic precursor of Formula V or Formula VI: with a macrocyclization reagent; wherein v, w, x, y, z, A, B, C, D, E, R 1 , R 2 , R 7 , R 8 , L 1 and L 2 are as defined for Formula (II); R 12 is -H when the macrocyclization reagent is a Cu reagent and R 12 is -H or alkyl when the macrocyclization reagent is a Ru reagent; and further wherein said contacting step results in a covalent linkage being formed between the alkyne and azide moiety in Formula III or Formula IV.
  • R 12 may be methyl when the macrocyclization reagent is a Ru reagent.
  • R 1 and R 2 are alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-.
  • both R 1 and R 2 are independently alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-.
  • At least one of A, B, C, D or E is an ⁇ , ⁇ -disubstituted amino acid.
  • B is an ⁇ , ⁇ -disubstituted amino acid.
  • at least one of A, B, C, D or E is 2-aminoisobutyric acid.
  • R 1 and R 2 are alkyl, unsubstituted or substituted with halo-. In another example, both R 1 and R 2 are independently alkyl, unsubstituted or substituted with halo-. In some embodiments, at least one of R 1 and R 2 is methyl. In other embodiments, R 1 and R 2 are methyl.
  • the macrocyclization reagent may be a Cu reagent or a Ru reagent.
  • the peptidomimetic precursor is purified prior to the contacting step.
  • the peptidomimetic macrocycle is purified after the contacting step.
  • the peptidomimetic macrocycle is refolded after the contacting step.
  • the method may be performed in solution, or, alternatively, the method may be performed on a solid support.
  • Also envisioned herein is performing the method of the invention in the presence of a target macromolecule that binds to the peptidomimetic precursor or peptidomimetic macrocycle under conditions that favor said binding.
  • the method is performed in the presence of a target macromolecule that binds preferentially to the peptidomimetic precursor or peptidomimetic macrocycle under conditions that favor said binding.
  • the method may also be applied to synthesize a library of peptidomimetic macrocycles.
  • the alkyne moiety of the peptidomimetic precursor of Formula V or Formula VI is a sidechain of an amino acid selected from the group consisting of L-propargylglycine, D-propargylglycine, (S)-2-amino-2-methyl-4-pentynoic acid, (R)-2-amino-2-methyl-4-pentynoic acid, (S)-2-amino-2-methyl-5-hexynoic acid, (R)-2-amino-2-methyl-5-hexynoic acid, (S)-2-amino-2-methyl-6-heptynoic acid, (R)-2-amino-2-methyl-6-heptynoic acid, (S)-2-amino-2-methyl-7-octynoic acid, (R)-2-amino-2-methyl-7-octynoic acid, (S)-2-amino-2-methyl-8-nonynoic acid, and (R)-2-amino-2-amino
  • the azide moiety of the peptidomimetic precursor of Formula V or Formula VI is a sidechain of an amino acid selected from the group consisting of ⁇ -azido-L-lysine, ⁇ -azido-D-lysine, ⁇ -azido- ⁇ -methyl-L-lysine, ⁇ -azido- ⁇ -methyl-D-lysine, ⁇ -azido-a-methyl-L-ornithine, and ⁇ -azido- ⁇ -methyl-D-ornithine.
  • x+y+z is 3, and and A, B and C are independently natural or non-natural amino acids. In other embodiments, x+y+z is 6, and and A, B and C are independently natural or non-natural amino acids.
  • [D] v and/or [E] w comprise additional peptidomimetic macrocycles or macrocyclic structures.
  • [D] v may have the formula: or wherein each A, C, D', and E' is independently a natural or non-natural amino acid;
  • [E] w has the formula: wherein the substituents are as defined in the preceding paragraph.
  • the contacting step is performed in a solvent selected from the group consisting of protic solvent, aqueous solvent, organic solvent, and mixtures thereof.
  • the solvent may be chosen from the group consisting of H 2 O, THF, THF/H 2 O, tBuOH/H 2 O, DMF, DIPEA, CH 3 CN or CH 2 Cl 2 , ClCH 2 CH 2 Cl or a mixture thereof.
  • the solvent may be a solvent which favors helix formation.
  • peptidomimetic macrocycles of the invention are made, for example, by chemical synthesis methods, such as described in Fields et al., Chapter 3 in Synthetic Peptides: A User's Guide, ed. Grant, W. H. Freeman & Co., New York, N. Y., 1992, p. 77 .
  • peptides are synthesized using the automated Merrifield techniques of solid phase synthesis with the amine protected by either tBoc or Fmoc chemistry using side chain protected amino acids on, for example, an automated peptide synthesizer (e.g., Applied Biosystems (Foster City, CA), Model 430A, 431, or 433).
  • One manner of producing the peptidomimetic precursors and peptidomimetic macrocycles described herein uses solid phase peptide synthesis (SPPS).
  • SPPS solid phase peptide synthesis
  • the C-terminal amino acid is attached to a cross-linked polystyrene resin via an acid labile bond with a linker molecule.
  • This resin is insoluble in the solvents used for synthesis, making it relatively simple and fast to wash away excess reagents and by-products.
  • the N-terminus is protected with the Fmoc group, which is stable in acid, but removable by base. Side chain functional groups are protected as necessary with base stable, acid labile groups.
  • peptidomimetic precursors are produced, for example, by conjoining individual synthetic peptides using native chemical ligation. Alternatively, the longer synthetic peptides are biosynthesized by well known recombinant DNA and protein expression techniques. Such techniques are provided in well-known standard manuals with detailed protocols.
  • To construct a gene encoding a peptidomimetic precursor of this invention the amino acid sequence is reverse translated to obtain a nucleic acid sequence encoding the amino acid sequence, preferably with codons that are optimum for the organism in which the gene is to be expressed.
  • a synthetic gene is made, typically by synthesizing oligonucleotides which encode the peptide and any regulatory elements, if necessary.
  • the synthetic gene is inserted in a suitable cloning vector and transfected into a host cell. The peptide is then expressed under suitable conditions appropriate for the selected expression system and host.
  • the peptide is purified and characterized by standard methods.
  • the peptidomimetic precursors are made, for example, in a high-throughput, combinatorial fashion using, for example, a high-throughput polychannel combinatorial synthesizer (e.g., Thuramed TETRAS multichannel peptide synthesizer from CreoSalus, Louisville, KY or Model Apex 396 multichannel peptide synthesizer from AAPPTEC, Inc., Louisville, KY).
  • a high-throughput polychannel combinatorial synthesizer e.g., Thuramed TETRAS multichannel peptide synthesizer from CreoSalus, Louisville, KY or Model Apex 396 multichannel peptide synthesizer from AAPPTEC, Inc., Louisville, KY.
  • each R 1 , R 2 , R 7 and R 8 is -H; each L 1 is -(CH 2 ) 4 -; and each L 2 is -(CH 2 )-.
  • R 1 , R 2 , R 7 , R 8 , L 1 and L 2 can be independently selected from the various structures disclosed herein.
  • Synthetic Scheme 1 describes the preparation of several compounds of the invention.
  • Ni(II) complexes of Schiff bases derived from the chiral auxiliary (S)-2-[N-(N'-benzylprolyl)amino]benzophenone (BPB) and amino acids such as glycine or alanine are prepared as described in Belokon et al. (1998), Tetrahedron Asymm. 9:4249-4252 .
  • the resulting complexes are subsequently reacted with alkylating reagents comprising an azido or alkynyl moiety to yield enantiomerically enriched compounds of the invention. If desired, the resulting compounds can be protected for use in peptide synthesis.
  • the peptidomimetic precursor contains an azide moiety and an alkyne moiety and is synthesized by solution-phase or solid-phase peptide synthesis (SPPS) using the commercially available amino acid N- ⁇ -Fmoc-L-propargylglycine and the N- ⁇ -Fmoc-protected forms of the amino acids (S)-2-amino-2-methyl-4-pentynoic acid, (S)-2-amino-6-heptynoic acid, (S)-2-amino-2-methyl-6-heptynoic acid, N-methyl- ⁇ -azido-L-lysine, and N-methyl- ⁇ -azido-D-lysine.
  • SPPS solution-phase or solid-phase peptide synthesis
  • the peptidomimetic precursor is then deprotected and cleaved from the solid-phase resin by standard conditions (e.g ., strong acid such as 95% TFA).
  • the peptidomimetic precursor is reacted as a crude mixture or is purified prior to reaction with a macrocyclization reagent such as a Cu(I) in organic or aqueous solutions ( Rostovtsev et al. (2002), Angew. Chem. Int. Ed. 41:2596-2599 ; Tornoe et al. (2002), J. Org. Chem. 67:3057-3064 ; Deiters et al. (2003), J. Am. Chem. Soc.
  • the triazole forming reaction is performed under conditions that favor ⁇ -helix formation.
  • the macrocyclization step is performed in a solvent chosen from the group consisting of H 2 O, THF, CH 3 CN, DMF , DIPEA, tBuOH or a mixture thereof.
  • the macrocyclization step is performed in DMF.
  • the macrocyclization step is performed in a buffered aqueous or partially aqueous solvent.
  • the peptidomimetic precursor contains an azide moiety and an alkyne moiety and is synthesized by solid-phase peptide synthesis (SPPS) using the commercially available amino acid N- ⁇ -Fmoc-L-propargylglycine and the N- ⁇ -Fmoc-protected forms of the amino acids (S)-2-amino-2-methyl-4-pentynoic acid, (S)-2-amino-6-heptynoic acid, (S)-2-amino-2-methyl-6-heptynoic acid, N-methyl- ⁇ -azido-L-lysine, and N-methyl- ⁇ -azido-D-lysine.
  • SPPS solid-phase peptide synthesis
  • the peptidomimetic precursor is reacted with a macrocyclization reagent such as a Cu(I) reagent on the resin as a crude mixture
  • a macrocyclization reagent such as a Cu(I) reagent
  • the resultant triazole-containing peptidomimetic macrocycle is then deprotected and cleaved from the solid-phase resin by standard conditions (e.g., strong acid such as 95% TFA).
  • the macrocyclization step is performed in a solvent chosen from the group consisting of CH 2 Cl 2 , ClCH 2 CH 2 Cl, DMF, THF, NMP, DIPEA, 2,6-lutidine, pyridine, DMSO, H 2 O or a mixture thereof.
  • the macrocyclization step is performed in a buffered aqueous or partially aqueous solvent.
  • the peptidomimetic precursor contains an azide moiety and an alkyne moiety and is synthesized by solution-phase or solid-phase peptide synthesis (SPPS) using the commercially available amino acid N- ⁇ -Fmoc-L-propargylglycine and the N- ⁇ -Fmoc-protected forms of the amino acids (S)-2-amino-2-methyl-4-pentynoic acid, (S)-2-amino-6-heptynoic acid, (S)-2-amino-2-methyl-6-heptynoic acid, N-methyl- ⁇ -azido-L-lysine, and N-methyl- ⁇ -azido-D-lysine.
  • SPPS solution-phase or solid-phase peptide synthesis
  • the peptidomimetic precursor is then deprotected and cleaved from the solid-phase resin by standard conditions (e.g ., strong acid such as 95% TFA).
  • the peptidomimetic precursor is reacted as a crude mixture or is purified prior to reaction with a macrocyclization reagent such as a Ru(II) reagents, for example Cp*RuCl(PPh 3 ) 2 or [Cp*RuCl] 4 ( Rasmussen et al. (2007), Org. Lett. 9:5337-5339 ; Zhang et al. (2005), J. Am. Chem. Soc. 127:15998-15999 ).
  • the macrocyclization step is performed in a solvent chosen from the group consisting of DMF, CH 3 CN and THF.
  • the peptidomimetic precursor contains an azide moiety and an alkyne moiety and is synthesized by solid-phase peptide synthesis (SPPS) using the commercially available amino acidN- ⁇ -Fmoc-L-propargylglycine and the N- ⁇ -Fmoc-protected forms of the amino acids (S)-2-amino-2-methyl-4-pentynoic acid, (S)-2-amino-6-heptynoic acid, (S)-2-amino-2-methyl-6-heptynoic acid, N-methyl- ⁇ -azido-L-lysine, and N-methyl- ⁇ -azido-D-lysine.
  • SPPS solid-phase peptide synthesis
  • the peptidomimetic precursor is reacted with a macrocyclization reagent such as a Ru(II) reagent on the resin as a crude mixture.
  • a macrocyclization reagent such as a Ru(II) reagent on the resin as a crude mixture.
  • the reagent can be Cp*RuCl(PPh 3 ) 2 or [Cp*RuCl] 4 ( Rasmussen et al. (2007), Org. Lett. 9:5337-5339 ; Zhang et al. (2005), J. Am. Chem. Soc. 127:15998-15999 ).
  • the macrocyclization step is performed in a solvent chosen from the group consisting of CH 2 Cl 2 , ClCH 2 CH 2 Cl, CH 3 CN, DMF, and THF.
  • the present invention contemplates the use of non-naturally-occurring amino acids and amino acid analogs in the synthesis of the peptidomimetic macrocycles described herein.
  • Any amino acid or amino acid analog amenable to the synthetic methods employed for the synthesis of stable triazole containing peptidomimetic macrocycles can be used in the present invention.
  • L-propargylglycine is contemplated as a useful amino acid in the present invention.
  • other alkyne-containing amino acids that contain a different amino acid side chain are also useful in the invention.
  • L-propargylglycine contains one methylene unit between the ⁇ -carbon of the amino acid and the alkyne of the amino acid side chain.
  • the invention also contemplates the use of amino acids with multiple methylene units between the ⁇ -carbon and the alkyne.
  • the azido-analogs of amino acids L-lysine, D-lysine, alpha-methyl-L-lysine, and alpha-methyl-D-lysine are contemplated as useful amino acids in the present invention.
  • other terminal azide amino acids that contain a different amino acid side chain are also useful in the invention.
  • the azido-analog of L-lysine contains four methylene units between the ⁇ -carbon of the amino acid and the terminal azide of the amino acid side chain.
  • the invention also contemplates the use of amino acids with fewer than or greater than four methylene units between the ⁇ -carbon and the terminal azide. Table 6 shows some amino acids useful in the preparation of peptidomimetic macrocycles of the invention.
  • the amino acids and amino acid analogs are of the D-configuration. In other embodiments they are of the L-configuration. In some embodiments, some of the amino acids and amino acid analogs contained in the peptidomimetic are of the D-configuration while some of the amino acids and amino acid analogs are of the L-configuration. In some embodiments the amino acid analogs are ⁇ , ⁇ -disubstituted, such as ⁇ -methyl-L-propargylglycine, ⁇ -methyl-D-propargylglycine, ⁇ -azido-alpha-methyl-L-lysine, and ⁇ -azido-alpha-methyl-D-lysine.
  • amino acid analogs are N-alkylated, e.g., N-methyl-L-propargylglycine, N-methyl-D-propargylglycine, N-methyl- ⁇ -azido-L-lysine, and N-methyl- ⁇ -azido-D-lysine.
  • the -NH moiety of the amino acid is protected using a protecting group, including without limitation -Fmoc and -Boc. In other embodiments, the amino acid is not protected prior to synthesis of the peptidomimetic macrocycle.
  • peptidomimetic macrocycles of Formula III are synthesized.
  • the following synthetic schemes describe the preparation of such compounds.
  • the illustrative schemes depict amino acid analogs derived from L-or D-cysteine, in which L 1 and L 3 are both -(CH 2 )-.
  • L 1 and L 3 can be independently selected from the various structures disclosed herein.
  • the symbols "[AA] m ", "[AA] n “, “[AA] o " represent a sequence of amide bond-linked moieties such as natural or unnatural amino acids.
  • each occurrence of "AA” is independent of any other occurrence of "AA", and a formula such as "[AA] m " encompasses, for example, sequences of non-identical amino acids as well as sequences of identical amino acids.
  • the peptidomimetic precursor contains two -SH moieties and is synthesized by solid-phase peptide synthesis (SPPS) using commercially available N- ⁇ -Fmoc amino acids such as N- ⁇ -Fmoc-S-trityl-L-cysteine or N- ⁇ -Fmoc-S-trityl-D-cysteine.
  • SPPS solid-phase peptide synthesis
  • Alpha-methylated versions of D-cysteine or L-cysteine are generated by known methods ( Seebach et al. (1996), Angew. Chem. Int. Ed. Engl.
  • N- ⁇ -Fmoc-S-trityl monomers 35:2708-2748 , and references therein) and then converted to the appropriately protected N- ⁇ -Fmoc-S-trityl monomers by known methods (" Bioorganic Chemistry: Peptides and Proteins", Oxford University Press, New York: 1998 , the entire contents of which are incorporated herein by reference).
  • the precursor peptidomimetic is then deprotected and cleaved from the solid-phase resin by standard conditions (e.g., strong acid such as 95% TFA).
  • the precursor peptidomimetic is reacted as a crude mixture or is purified prior to reaction with X-L 2 -Y in organic or aqueous solutions.
  • the alkylation reaction is performed under dilute conditions (i.e.
  • the alkylation reaction is performed in organic solutions such as liquid NH 3 ( Mosberg et al. (1985), J. Am.Chem. Soc. 107:2986-2987 ; Szewczuk et al. (1992), Int. J. Peptide Protein Res. 40 :233-242 ), NH 3 /MeOH, or NH 3 /DMF ( Or et al. (1991), J. Org. Chem. 56:3146-3149 ).
  • the alkylation is performed in an aqueous solution such as 6M guanidinium HCL, pH 8 ( Brunel et al. (2005), Chem. Commun. (20):2552-2554 ).
  • the solvent used for the alkylation reaction is DMF or dichloroethane.
  • the precursor peptidomimetic contains two or more -SH moieties, of which two are specially protected to allow their selective deprotection and subsequent alkylation for macrocycle formation.
  • the precursor peptidomimetic is synthesized by solid-phase peptide synthesis (SPPS) using commercially available N- ⁇ -Fmoc amino acids such as N- ⁇ -Fmoc-S- p -methoxytrityl-L-cysteine or N- ⁇ -Fmoc-S- p- methoxytrityl-D-cysteine.
  • SPPS solid-phase peptide synthesis
  • Alpha-methylated versions of D-cysteine or L-cysteine are generated by known methods ( Seebach et al. (1996), Angew. Chem. Int.
  • the reaction takes place in the presence of a hindered base such as diisopropylethylamine.
  • a hindered base such as diisopropylethylamine.
  • the alkylation reaction is performed in organic solutions such as liquid NH 3 ( Mosberg et al. (1985), J. Am.Chem. Soc. 107:2986-2987 ; Szewczuk et al. (1992), Int. J. Peptide Protein Res. 40 :233-242 ), NH 3 /MeOH or NH 3 /DMF ( Or et al. (1991), J. Org. Chem. 56:3146-3149 ).
  • the alkylation reaction is performed in DMF or dichloroethane.
  • the peptidomimetic macrocycle is then deprotected and cleaved from the solid-phase resin by standard conditions (e.g., strong acid such as 95% TFA).
  • the peptidomimetic precursor contains two or more -SH moieties, of which two are specially protected to allow their selective deprotection and subsequent alkylation for macrocycle formation.
  • the peptidomimetic precursor is synthesized by solid-phase peptide synthesis (SPPS) using commercially available N- ⁇ -Fmoc amino acids such as N- ⁇ -Fmoc-S- p -methoxytrityl-L-cysteine, N- ⁇ -Fmoc-S- p- methoxytrityl-D-cysteine, N- ⁇ -Fmoc-S-S-t-butyl-L-cysteine, and N- ⁇ -Fmoc-S-S-t-butyl-D-cysteine.
  • SPPS solid-phase peptide synthesis
  • Alpha-methylated versions of D-cysteine or L-cysteine are generated by known methods ( Seebach et al. (1996), Angew. Chem. Int. Ed. Engl. 35:2708-2748 , and references therein) and then converted to the appropriately protected N- ⁇ -Fmoc-S- p -methoxytrityl or N- ⁇ -Fmoc-S-S-t-butyl monomers by known methods ( Bioorganic Chemistry: Peptides and Proteins, Oxford University Press, New York: 1998 , the entire contents of which are incorporated herein by reference).
  • the S-S-tButyl protecting group of the peptidomimetic precursor is selectively cleaved by known conditions (e.g., 20% 2-mercaptoethanol in DMF, reference: Gauß et al. (2005), J. Comb. Chem. 7:174-177 ).
  • the precursor peptidomimetic is then reacted on the resin with a molar excess of X-L 2 -Y in an organic solution. For example, the reaction takes place in the presence of a hindered base such as diisopropylethylamine.
  • the Mmt protecting group of the peptidomimetic precursor is then selectively cleaved by standard conditions (e.g., mild acid such as 1% TFA in DCM).
  • the peptidomimetic precursor is then cyclized on the resin by treatment with a hindered base in organic solutions.
  • the alkylation reaction is performed in organic solutions such as NH 3 /MeOH or NH 3 /DMF ( Or et al. (1991), J. Org. Chem. 56:3146-3149 ).
  • the peptidomimetic macrocycle is then deprotected and cleaved from the solid-phase resin by standard conditions (e.g., strong acid such as 95% TFA).
  • the peptidomimetic precursor contains two L-cysteine moieties.
  • the peptidomimetic precursor is synthesized by known biological expression systems in living cells or by known in vitro, cell-free, expression methods.
  • the precursor peptidomimetic is reacted as a crude mixture or is purified prior to reaction with X-L2-Y in organic or aqueous solutions.
  • the alkylation reaction is performed under dilute conditions (i.e. 0.15 mmol/L) to favor macrocyclization and to avoid polymerization.
  • the alkylation reaction is performed in organic solutions such as liquid NH 3 ( Mosberg et al. (1985), J. Am.Chem. Soc.
  • the alkylation is performed in an aqueous solution such as 6M guanidinium HCL, pH 8 ( Brunel et al. (2005), Chem. Commun. (20):2552-2554 ). In other embodiments, the alkylation is performed in DMF or dichloroethane.
  • the alkylation is performed in non-denaturing aqueous solutions, and in yet another embodiment the alkylation is performed under conditions that favor ⁇ -helical structure formation. In yet another embodiment, the alkylation is performed under conditions that favor the binding of the precursor peptidomimetic to another protein, so as to induce the formation of the bound ⁇ -helical conformation during the alkylation.
  • X and Y are envisioned which are suitable for reacting with thiol groups.
  • each X or Y is independently be selected from the general category shown in Table 5.
  • X and Y are halides such as -Cl, -Br or -I.
  • Any of the macrocycle-forming linkers described herein may be used in any combination with any of the sequences shown in Tables 1-4 and also with any of the R- substituents indicated herein.
  • TABLE 7 Examples of Reactive Groups Capable of Reacting with Thiol Groups and Resulting Linkages (1) X or Y (2) Resulting Covalent Linkage (3) acrylamide (4) Thioether (5) halide (e.g.
  • Table 8 shows exemplary macrocycles of the invention.
  • the present invention contemplates the use of both naturally-occurring and non-naturally-occurring amino acids and amino acid analogs in the synthesis of the peptidomimetic macrocycles of Formula (III).
  • Any amino acid or amino acid analog amenable to the synthetic methods employed for the synthesis of stable bis-sulfhydryl containing peptidomimetic macrocycles can be used in the present invention.
  • cysteine is contemplated as a useful amino acid in the present invention.
  • sulfur containing amino acids other than cysteine that contain a different amino acid side chain are also useful.
  • cysteine contains one methylene unit between the ⁇ -carbon of the amino acid and the terminal -SH of the amino acid side chain.
  • the invention also contemplates the use of amino acids with multiple methylene units between the ⁇ -carbon and the terminal -SH.
  • Non-limiting examples include ⁇ -methyl-L-homocysteine and ⁇ -methyl-D-homocysteine.
  • the amino acids and amino acid analogs are of the D-configuration. In other embodiments they are of the L- configuration.
  • some of the amino acids and amino acid analogs contained in the peptidomimetic are of the D- configuration while some of the amino acids and amino acid analogs are of the L- configuration.
  • the amino acid analogs are ⁇ , ⁇ -disubstituted, such as ⁇ -methyl-L-cysteine and ⁇ -methyl-D-cysteine.
  • the invention includes macrocycles in which macrocycle-forming linkers are used to link two or more -SH moieties in the peptidomimetic precursors to form the peptidomimetic macrocycles of the invention.
  • the macrocycle-forming linkers impart conformational rigidity, increased metabolic stability and/or increased cell penetrability.
  • the macrocycle-forming linkages stabilize the ⁇ -helical secondary structure of the peptidomimetic macrocyles.
  • the macrocycle-forming linkers are of the formula X-L 2 -Y, wherein both X and Y are the same or different moieties, as defined above.
  • Both X and Y have the chemical characteristics that allow one macrocycle-forming linker-L 2 - to bis alkylate the bis-sulfhydryl containing peptidomimetic precursor.
  • the linker-L 2 - includes alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, or heterocycloarylene, or -R 4 -K-R 4 -, all of which can be optionally substituted with an R 5 group, as defined above.
  • one to three carbon atoms within the macrocycle-forming linkers-L 2 -, other than the carbons attached to the -SH of the sulfhydryl containing amino acid, are optionally substituted with a heteroatom such as N, S or O.
  • the L 2 component of the macrocycle-forming linker X-L 2 -Y may be varied in length depending on, among other things, the distance between the positions of the two amino acid analogs used to form the peptidomimetic macrocycle. Furthermore, as the lengths of L 1 and/or L 3 components of the macrocycle-forming linker are varied, the length of L 2 can also be varied in order to create a linker of appropriate overall length for forming a stable peptidomimetic macrocycle. For example, if the amino acid analogs used are varied by adding an additional methylene unit to each of L 1 and L 3 , the length of L 2 are decreased in length by the equivalent of approximately two methylene units to compensate for the increased lengths of L 1 and L 3 .
  • L 2 is an alkylene group of the formula -(CH 2 ) n -, where n is an integer between about 1 and about 15. For example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In other embodiments, L 2 is an alkenylene group. In still other embodiments, L 2 is an aryl group.
  • Table 9 shows additional embodiments of X-L 2 -Y groups.
  • Each X and Y in this table is, for example, independently Cl-, Br- or I-.
  • aminoacid precursors are used containing an additional substituent R- at the alpha position.
  • Such aminoacids are incorporated into the macrocycle precursor at the desired positions, which may be at the positions where the crosslinker is substituted or, alternatively, elsewhere in the sequence of the macrocycle precursor. Cyclization of the precursor is then effected according to the indicated method.
  • the properties of the peptidomimetic macrocycles of the invention are assayed, for example, by using the methods described below.
  • polypeptides with ⁇ -helical domains will reach a dynamic equilibrium between random coil structures and ⁇ -helical structures, often expressed as a "percent helicity".
  • unmodified pro-apoptotic BH3 domains are predominantly random coils in solution, with ⁇ -helical content usually under 25%.
  • Peptidomimetic macrocycles with optimized linkers possess, for example, an alpha-helicity that is at least two-fold greater than that of a corresponding uncrosslinked polypeptide.
  • macrocycles of the invention will possess an alpha-helicity of greater than 50%.
  • aqueous solution e.g. 50 mM potassium phosphate solution at pH 7, or distilled H 2 O, to concentrations of 25-50 ⁇ M.
  • Circular dichroism (CD) spectra are obtained on a spectropolarimeter (e.g., Jasco J-710) using standard measurement parameters (e.g. temperature, 20°C; wavelength, 190-260 nm; step resolution, 0.5 nm; speed, 20 nm/sec; accumulations, 10; response, 1 sec; bandwidth, 1 nm; path length, 0.1 cm).
  • the ⁇ -helical content of each peptide is calculated by dividing the mean residue ellipticity (e.g. [ ⁇ ]222obs) by the reported value for a model helical decapeptide ( Yang et al. (1986), Methods Enzymol. 130:208 )).
  • a peptidomimetic macrocycle of the invention comprising a secondary structure such as an ⁇ -helix exhibits, for example, a higher melting temperature than a corresponding uncrosslinked polypeptide.
  • peptidomimetic macrocycles of the invention exhibit Tm of > 60°C representing a highly stable structure in aqueous solutions.
  • Tm is determined by measuring the change in ellipticity over a temperature range (e.g.
  • spectropolarimeter e.g., Jasco J-710
  • standard parameters e.g. wavelength 222nm; step resolution, 0.5 nm; speed, 20 nm/sec; accumulations, 10; response, 1 sec; bandwidth, 1 nm; temperature increase rate: 1°C/min; path length, 0.1 cm.
  • the amide bond of the peptide backbone is susceptible to hydrolysis by proteases, thereby rendering peptidic compounds vulnerable to rapid degradation in vivo. Peptide helix formation, however, typically buries the amide backbone and therefore may shield it from proteolytic cleavage.
  • the peptidomimetic macrocycles of the present invention may be subjected to in vitro trypsin proteolysis to assess for any change in degradation rate compared to a corresponding uncrosslinked polypeptide.
  • the peptidomimetic macrocycle and a corresponding uncrosslinked polypeptide are incubated with trypsin agarose and the reactions quenched at various time points by centrifugation and subsequent HPLC injection to quantitate the residual substrate by ultraviolet absorption at 280 nm.
  • the peptidomimetic macrocycle and peptidomimetic precursor (5 mcg) are incubated with trypsin agarose (Pierce) (S/E ⁇ 125) for 0, 10, 20, 90, and 180 minutes. Reactions are quenched by tabletop centrifugation at high speed; remaining substrate in the isolated supernatant is quantified by HPLC-based peak detection at 280 nm.
  • Peptidomimetic macrocycles with optimized linkers possess, for example, an ex vivo half-life that is at least two-fold greater than that of a corresponding uncrosslinked polypeptide, and possess an ex vivo half-life of 12 hours or more.
  • a variety of assays may be used.
  • a peptidomimetic macrocycle and/or a corresponding uncrosslinked polypeptide (2 mcg) are each incubated with fresh mouse, rat and/or human serum (e.g. 1-2 mL) at 37°C for 0, 1, 2, 4, 8, and 24 hours. Samples of differing macrocycle concentration may be prepared by serial dilution with serum.
  • the samples are extracted by transferring 100 ⁇ l of sera to 2 ml centrifuge tubes followed by the addition of 10 ⁇ L of 50 % formic acid and 500 ⁇ L acetonitrile and centrifugation at 14,000 RPM for 10 min at 4 ⁇ 2°C. The supernatants are then transferred to fresh 2 ml tubes and evaporated on Turbovap under N 2 ⁇ 10 psi, 37°C. The samples are reconstituted in 100 ⁇ L of 50:50 acetonitrile:water and submitted to LC-MS/MS analysis. Equivalent or similar procedures for testing ex vivo stability are known and may be used to determine stability of macrocycles in serum.
  • FPA fluorescence polarization assay
  • fluoresceinated peptidomimetic macrocycles (25 nM) are incubated with the acceptor protein (25- 1000nM) in binding buffer (140mM NaCl, 50 mM Tris-HCL, pH 7.4) for 30 minutes at room temperature. Binding activity ismeasured, for example, by fluorescence polarization on a luminescence spectrophotometer (e.g. Perkin-Elmer LS50B). Kd values may be determined by nonlinear regression analysis using, for example, Graphpad Prism software (GraphPad Software, Inc., San Diego, CA).
  • a peptidomimetic macrocycle of the invention shows, in some instances, similar or lower Kd than a corresponding uncrosslinked polypeptide.
  • Acceptor proteins for BH3-peptides such as BCL-2, BCL-X L , BAX or MCL1 may, for example, be used in this assay. Additional methods to perform such assays are described in the Example section below.
  • a fluorescence polarization assay utilizing a fluoresceinated peptidomimetic macrocycle derived from a peptidomimetic precursor sequence is used, for example.
  • the FPA technique measures the molecular orientation and mobility using polarized light and fluorescent tracer. When excited with polarized light, fluorescent tracers (e.g., FITC) attached to molecules with high apparent molecular weights (e.g.
  • FITC-labeled peptides bound to a large protein emit higher levels of polarized fluorescence due to their slower rates of rotation as compared to fluorescent tracers attached to smaller molecules (e.g. FITC-labeled peptides that are free in solution).
  • a compound that antagonizes the interaction between the fluoresceinated peptidomimetic macrocycle and an acceptor protein will be detected in a competitive binding FPA experiment.
  • putative antagonist compounds (1 nM to 1 mM) and a fluoresceinated peptidomimetic macrocycle (25 nM) are incubated with the acceptor protein (50 nM) in binding buffer (140mM NaCl, 50 mM Tris-HCL, pH 7.4) for 30 minutes at room temperature.
  • Antagonist binding activity ismeasured, for example, by fluorescence polarization on a luminescence spectrophotometer (e.g. Perkin-Elmer LS50B).
  • Kd values may be determined by nonlinear regression analysis using, for example, Graphpad Prism software (GraphPad Software, Inc., San Diego, CA).
  • Any class of molecule such as small organic molecules, peptides, oligonucleotides or proteins can be examined as putative antagonists in this assay.
  • Acceptor proteins for BH3-peptides such as BCL2, BCL-XL, BAX or MCL1 can be used in this assay. Additional methods to perform such assays are described in the Example section below.
  • FITC-labeled fluoresceinated
  • biotinylated compounds for 4 hrs in the absence of serum, followed by serum replacement and further incubation that ranges from 4-18 hrs.
  • Opti-MEM Invitrogen
  • Cells are then pelleted and incubated in lysis buffer (50mM Tris [pH 7.6], 150 mM NaCl, 1% CHAPS and protease inhibitor cocktail) for 10 minutes at 4°C.
  • 1 % NP-40 or Triton X-100 may be used instead of CHAPS. Extracts are centrifuged at 14,000 rpm for 15 minutes and supernatants collected and incubated with 10 ⁇ l goat anti-FITC antibody or streptavidin-coated beads for 2 hrs, rotating at 4°C followed by further 2 hrs incubation at 4°C with protein A/G Sepharose (50 ⁇ l of 50% bead slurry).). No secondary step is necessary if using streptavidin beads to pull down biotinylated compounds.
  • FITC-labeled or biotinylated compounds are incubated with cell lysates, prepared as described above, for 2 hrs, rotating at 4°C followed by incubation with 10 ⁇ l goat anti-FITC antibody or streptavidin-coated beads for 2 hrs, rotating at 4°C followed by further 2 hrs incubation at 4°C with protein A/G Sepharose (50 ⁇ l of 50% bead slurry), no secondary step is necessary if using streptavidin beads to pull down biotinylated compounds.After quick centrifugation, the pellets may be washed in lysis buffer containing increasing salt concentration (e.g., 150, 300, 500 mM of NaCl).
  • salt concentration e.g. 150, 300, 500 mM of NaCl
  • the beads may be then re-equilibrated at 150 mM NaCl before addition of SDS-containing sample buffer and boiling.
  • the beads and cell lysates may be electrophoresed using 4%-12% gradient Bis-Tris gels followed by transfer into Immobilon-P membranes. After blocking, blots may be incubated with an antibody that detects FITC or biotin, respectively and also with one or more antibodies that detect proteins that bind to the peptidomimetic macrocycle, including BCL2, MCL1, BCL-XL, A1, BAX, and BAK.
  • the lysate blots are also probed with anti-Hsc-70 for loading control.
  • the gel may be silver stained to detect proteins that come down specifically with FITC-labeled or biotinylated compounds.
  • a peptidomimetic macrocycle is, for example, more cell permeable compared to a corresponding uncrosslinked polypeptide. In some embodiments, the peptidomimetic macrocycles are more cell permeable than a corresponding uncrosslinked polypeptides.
  • Peptidomimetic macrocycles with optimized linkers possess, for example, cell penetrability that is at least two-fold greater than a corresponding uncrosslinked polypeptide, and often 20% or more of the applied peptidomimetic macrocycle will be observed to have penetrated the cell after 4 hours.
  • intact cells are incubated with fluoresceinated peptidomimetic macrocycles or corresponding uncrosslinked polypeptides (10 ⁇ M) for 4 hrs in serum free media at 37°C, washed twice with media and incubated with trypsin (0.25%) for 10 min at 37°C.
  • the cells are washed again and resuspended in PBS.
  • Cellular fluorescence is analyzed, for example, by using either a FACSCalibur flow cytometer or Cellomics' KineticScan ® HCS Reader. Additional methods of quantitating cellular penetration may be used. A particular method is described in more details in the Examples provided.
  • EC 50 refers to the half maximal effective concentration, which is the concentration of peptidomimetic macrocycle at which 50% the population is viable.
  • assays that measure Annexin V and caspase activation are optionally used to assess whether the peptidomimetic macrocycles kill cells by activating the apoptotic machinery.
  • the Cell Titer-glo assay is used which determines cell viability as a function of intracellular ATP concentration.
  • the compounds are, for example,administered to mice and/or rats by IV, IP, SC, PO or inhalation routes at concentrations ranging from 0.1 to 50 mg/kg and blood specimens withdrawn at 0', 5', 15', 30', 1 hr, 4 hrs, 8 hrs, 12 hrs, 24 hrs and 48 hrs post-injection. Levels of intact compound in 25 ⁇ L of fresh serum are then measured by LC-MS/MS as described herein.
  • the compounds are, for example, given alone (IP, IV, SC, PO, by inhalation or nasal routes) or in combination with sub-optimal doses of relevant chemotherapy (e.g., cyclophosphamide, doxorubicin, etoposide).
  • relevant chemotherapy e.g., cyclophosphamide, doxorubicin, etoposide.
  • 5 x 10 6 SEMK2 cells (established from the bone marrow of a patient with acute lymphoblastic leukemia) that stably express luciferase are injected by tail vein in NOD-SCID, SCID-beige or NOD.IL2rg KO mice 3 hrs after they have been subjected to total body irradiation.
  • Non-radiated mice may also be used for these studies. If left untreated, this form of leukemia is fatal in 3 weeks in this model. The leukemia is readily monitored, for example, by injecting the mice with D-luciferin (60 mg/kg) and imaging the anesthetized animals (e.g ., Xenogen In Vivo Imaging System, Caliper Life Sciences, Hopkinton, MA). Total body bioluminescence is quantified by integration of photonic flux (photons/sec) by Living Image Software (Caliper Life Sciences, Hopkinton, MA).
  • Peptidomimetic macrocycles alone or in combination with sub-optimal doses of relevant chemotherapeutics agents are, for example, administered to leukemic mice (8-10 days after injection/day 1 of experiment, in bioluminescence range of 14-16) by tail vein or IP routes at doses ranging from 0.1mg/kg to 50 mg/kg for 7 to 21 days.
  • the mice are imaged throughout the experiment every other day and survival monitored daily for the duration of the experiment.
  • Expired mice are optionally subjected to necropsy at the end of the experiment.
  • Another animal model is implantation into NOD-SCID mice of DoHH2, a cell line derived from human follicular lymphoma, that stably expresses luciferase. These in vivo tests optionally generate preliminary pharmacokinetic, pharmacodynamic and toxicology data.
  • peptidomimetic macrocycles of the invention are selected and separated in treatment and one or more control groups, wherein the treatment group is administered a peptidomimetic macrocycle of the invention, while the control groups receive a placebo, a known anti-cancer drug, or the standard of care.
  • the treatment safety and efficacy of the peptidomimetic macrocycles of the invention can thus be evaluated by performing comparisons of the patient groups with respect to factors such as survival and quality-of-life.
  • the patient group treated with a peptidomimetic macrocyle show improved long-term survival compared to a patient control group treated with a placebo or the standard of care.
  • Methods of administration include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intracerebral, intravaginal, transdermal, rectal, by inhalation, or topical by application to ears, nose, eyes, or skin.
  • the peptidomimetic macrocycles of the invention also include pharmaceutically acceptable derivatives or prodrugs thereof.
  • a "pharmaceutically acceptable derivative” means any pharmaceutically acceptable salt, ester, salt of an ester, pro-drug or other derivative of a compound of this invention which, upon administration to a recipient, is capable of providing (directly or indirectly) a compound of this invention.
  • pharmaceutically acceptable derivatives may increase the bioavailability of the compounds of the invention when administered to a mammal (e.g ., by increasing absorption into the blood of an orally administered compound) or which increases delivery of the active compound to a biological compartment (e.g ., the brain or lymphatic system) relative to the parent species.
  • Some pharmaceutically acceptable derivatives include a chemical group which increases aqueous solubility or active transport across the gastrointestinal mucosa.
  • the peptidomimetic macrocycles of the invention are modified by covalently or non-covalently joining appropriate functional groups to enhance selective biological properties.
  • modifications include those which increase biological penetration into a given biological compartment (e.g., blood, lymphatic system, central nervous system), increase oral availability, increase solubility to allow administration by injection, alter metabolism, and alter rate of excretion.
  • Pharmaceutically acceptable salts of the compounds of this invention include those derived from pharmaceutically acceptable inorganic and organic acids and bases.
  • suitable acid salts include acetate, adipate, benzoate, benzenesulfonate, butyrate, citrate, digluconate, dodecylsulfate, formate, fumarate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, palmoate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, tosylate and undecanoate.
  • Salts derived from appropriate bases include alkali metal (e.g., sodium), alkaline earth metal (e.g., magnesium), ammonium and N
  • pharmaceutically acceptable carriers include either solid or liquid carriers.
  • Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules.
  • a solid carrier can be one or more substances, which also acts as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material. Details on techniques for formulation and administration are well described in the scientific and patent literature, see, e.g., the latest edition of Remington's Pharmaceutical Sciences, Maack Publishing Co, Easton PA .
  • the carrier is a finely divided solid, which is in a mixture with the finely divided active component.
  • the active component is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.
  • Suitable solid excipients are carbohydrate or protein fillers include, but are not limited to sugars, including dextrose, lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; and gums including arabic and tragacanth; as well as proteins such as gelatin and collagen.
  • disintegrating or solubilizing agents are added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
  • Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water/propylene glycol solutions.
  • liquid preparations can be formulated in solution in aqueous polyethylene glycol solution.
  • parenteral refers modes of administration including intravenous, intraarterial, intramuscular, intraperitoneal, intrasternal, and subcutaneous.
  • the pharmaceutical preparation is preferably in unit dosage form.
  • the preparation is subdivided into unit doses containing appropriate quantities of the active component.
  • the unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules.
  • the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.
  • compositions of this invention comprise a combination of a peptidomimetic macrocycle and one or more additional therapeutic or prophylactic agents
  • both the compound and the additional agent should be present at dosage levels of between about 1 to 100%, and more preferably between about 5 to 95% of the dosage normally administered in a monotherapy regimen.
  • the additional agents are administered separately, as part of a multiple dose regimen, from the compounds of this invention.
  • those agents are part of a single dosage form, mixed together with the compounds of this invention in a single composition.
  • the present invention provides novel peptidomimetic macrocycles that are useful in competitive binding assays to identify agents which bind to the natural ligand(s) of the proteins or peptides upon which the peptidomimetic macrocycles are modeled.
  • labeled peptidomimetic macrocycles based on BH3 can be used in a BCL-X L binding assay along with small molecules that competitively bind to BCL-X L .
  • Competitive binding studies allow for rapid in vitro evaluation and determination of drug candidates specific for the BH3/BCL-X L system.
  • the invention further provides for the generation of antibodies against the peptidomimetic macrocycles.
  • these antibodies specifically bind both the peptidomimetic macrocycle and the BH3 peptidomimetic precursors upon which the peptidomimetic macrocycles are derived.
  • Such antibodies for example, disrupt the BH3/BCL-XL systems, respectively.
  • the present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant (e.g., insuffcient or excessive) BCL-2 family member expression or activity (e.g., extrinsic or intrinsic apoptotic pathway abnormalities).
  • a disorder associated with aberrant e.g., insuffcient or excessive
  • BCL-2 family member expression or activity e.g., extrinsic or intrinsic apoptotic pathway abnormalities.
  • some BCL-2 type disorders are caused, at least in part, by an abnormal level of one or more BCL-2 family members (e.g., over or under expression), or by the presence of one or more BCL-2 family members exhibiting abnormal activity.
  • the reduction in the level and/or activity of the BCL-2 family member or the enhancement of the level and/or activity of the BCL-2 family member is used, for example, to ameliorate or reduce the adverse symptoms of the disorder.
  • the compounds of the invention are used to treat disorders associated with expression or overexpression of Mcl-1.
  • Mcl-1 has been shown to be expressed in many tissues and neoplastic cell lines and is thought to participate in the development of malignancies ( Thallinger et al. (2004) Clin. Cancer Res. 10:4185-4191 ).
  • the peptidomimetic macrocycles of the invention may be used for the treatment of such malignancies.
  • the disorder being treated is differentially responsive to the peptidomimetic macrocycles of the invention.
  • the cancer is treated with a BIM peptidomimetic macrocycle and is at least 2-fold less sensitive to treatment using a BID polypeptide (such as a BID peptidomimetic macrocycle or uncrosslinked polypeptide) as measured in an in vitro cell viability assay.
  • a BID polypeptide such as a BID peptidomimetic macrocycle or uncrosslinked polypeptide
  • the cancer is at least 5-fold less sensitive to treatment using a BID polypeptide as measured in an in vitro cell viability assay.
  • the cancer is at least 8-fold less sensitive to treatment using a BID polypeptide as measured in an in vitro cell viability assay.
  • the cancer is treated with a BID peptidomimetic macrocycle and is at least 2-fold less sensitive to treatment using a BIM polypeptide (such as a BIM peptidomimetic macrocycle or uncrosslinked polypeptide) as measured in an in vitro cell viability assay.
  • a BIM polypeptide such as a BIM peptidomimetic macrocycle or uncrosslinked polypeptide
  • the cancer is at least 5-fold less sensitive to treatment using a BIM polypeptide as measured in an in vitro cell viability assay.
  • the cancer is at least 8-fold less sensitive to treatment using a BIM polypeptide as measured in an in vitro cell viability assay.
  • a method of treating a human patient comprising performing an assay to evaluate the levels of a BCL-family protein and administering to the patient a peptidomimetic macrocycle if an abberant or irregular level of expression of the BCL-family protein is detected.
  • BCL-family proteins include, for example, BCL-2, BCL-XL, MCL-1, Bfl1/A1, BOO/DIVA, NRH/NR13, BAX, BAD, BAK, BOK, BIK, PUMA, BIM, BMF, BLK, BNIP3,HRK, NIX, SPIKE, and Noxa.
  • a method of treating a human patient comprising performing an assay to evaluate the levels of BCL-2 in the patient and administering to the patient a peptidomimetic macrocycle if an abberant or irregular level of expression of BCL-2 is detected.
  • a method of treating a human patient comprising performing an assay to evaluate the levels of BCL-XL in the patient and administering to the patient a peptidomimetic macrocycle if an abberant or irregular level of expression of BCL-XL is detected.
  • a method of treating a human patient comprising performing an assay to evaluate the levels of MCL-1 in the patient and administering to the patient a peptidomimetic macrocycle if an abberant or irregular level of expression of MCL-1 is detected.
  • a method of treating a human patient comprising performing an assay to evaluate the levels of BAX in the patient and administering to the patient a peptidomimetic macrocycle if an abberant or irregular level of expression of BAX is detected.
  • a method of treating a human patient comprising performing an assay to evaluate the levels of BAD in the patient and administering to the patient a peptidomimetic macrocycle if an abberant or irregular level of expression of BAD is detected.
  • a method of treating a human patient comprising performing an assay to evaluate the levels of BAK in the patient and administering to the patient a peptidomimetic macrocycle if an abberant or irregular level of expression of BAK is detected.
  • a method of treating a human patient comprising performing an assay to evaluate the levels of PUMA in the patient and administering to the patient a peptidomimetic macrocycle if an abberant or irregular level of expression of PUMA is detected.
  • a method of treating a human patient comprising performing an assay to evaluate the levels of Noxa in the patient and administering to the patient a peptidomimetic macrocycle if an abberant or irregular level of expression of Noxa is detected.
  • a method of treating a human patient comprising performing an assay to evaluate the levels of Noxa in the patient and administering to the patient a peptidomimetic macrocycle if an abberant or irregular level of expression of Noxa is detected.
  • a method of treating a human patient comprising performing an assay to evaluate the levels of Bfl1/A1 in the patient and administering to the patient a peptidomimetic macrocycle if an abberant or irregular level of expression of Bfl1/A1 is detected.
  • a method of treating a human patient comprising performing an assay to evaluate the levels of BOO/DIVA in the patient and administering to the patient a peptidomimetic macrocycle if an abberant or irregular level of expression of BOO/DIVA is detected.
  • a method of treating a human patient comprising performing an assay to evaluate the levels of NRH/NR13 in the patient and administering to the patient a peptidomimetic macrocycle if an abberant or irregular level of expression of NRH/NR13 is detected.
  • a method of treating a human patient comprising performing an assay to evaluate the levels of BOK in the patient and administering to the patient a peptidomimetic macrocycle if an abberant or irregular level of expression of BOK is detected.
  • a method of treating a human patient comprising performing an assay to evaluate the levels of BIK in the patient and administering to the patient a peptidomimetic macrocycle if an abberant or irregular level of expression of BIK is detected.
  • a method of treating a human patient comprising performing an assay to evaluate the levels of BMF in the patient and administering to the patient a peptidomimetic macrocycle if an abberant or irregular level of expression of BMF is detected.
  • a method of treating a human patient comprising performing an assay to evaluate the levels of BLK in the patient and administering to the patient a peptidomimetic macrocycle if an abberant or irregular level of expression of BLK is detected.
  • a method of treating a human patient comprising performing an assay to evaluate the levels of BNIP3 in the patient and administering to the patient a peptidomimetic macrocycle if an abberant or irregular level of expression of BNIP3 is detected.
  • a method of treating a human patient comprising performing an assay to evaluate the levels of HRK in the patient and administering to the patient a peptidomimetic macrocycle if an abberant or irregular level of expression of HRK is detected.
  • a method of treating a human patient comprising performing an assay to evaluate the levels of Nix in the patient and administering to the patient a peptidomimetic macrocycle if an abberant or irregular level of expression of Nix is detected.
  • a method of treating a human patient comprising performing an assay to evaluate the levels of SPIKE in the patient and administering to the patient a peptidomimetic macrocycle if an abberant or irregular level of expression of SPIKE is detected.
  • treatment is defined as the application or administration of a therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disease, a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease or the predisposition toward disease.
  • the peptidomimetics macrocycles of the invention is used to treat, prevent, and/or diagnose cancers and neoplastic conditions.
  • cancer hyperproliferative and neoplastic
  • hyperproliferative and neoplastic disease states may be categorized as pathologic, i.e., characterizing or constituting a disease state, or may be categorized as non-pathologic, i.e. , a deviation from normal but not associated with a disease state.
  • metastatic tumor can arise from a multitude of primary tumor types, including but not limited to those of breast, lung, liver, colon and ovarian origin.
  • Primary tumor types including but not limited to those of breast, lung, liver, colon and ovarian origin.
  • Primary tumor types including but not limited to those of breast, lung, liver, colon and ovarian origin.
  • Primary tumor types including but not limited to those of breast, lung, liver, colon and ovarian origin.
  • Primary tumor growth including but not limited to those of breast, lung, liver, colon and ovarian origin.
  • “Pathologic hyperproliferative" cells occur in disease states characterized by malignant tumor growth.
  • non-pathologic hyperproliferative cells include proliferation of cells associated with wound repair.
  • cellular proliferative and/or differentiative disorders include cancer, e.g., carcinoma, sarcoma, or metastatic disorders.
  • the peptidomimetics macrocycles are novel therapeutic agents for controlling breast cancer, ovarian cancer, colon cancer, lung cancer, metastasis
  • cancers or neoplastic conditions include, but are not limited to, a fibrosarcoma, myosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, gastric cancer, esophageal cancer, rectal cancer, pancreatic cancer, ovarian cancer, prostate cancer, uterine cancer, cancer of the head and neck, skin cancer, brain cancer, squamous cell carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile
  • proliferative disorders examples include hematopoietic neoplastic disorders.
  • hematopoietic neoplastic disorders includes diseases involving hyperplastic/neoplastic cells of hematopoietic origin, e.g ., arising from myeloid, lymphoid or erythroid lineages, or precursor cells thereof.
  • the diseases arise from poorly differentiated acute leukemias, e.g ., erythroblastic leukemia and acute megakaryoblastic leukemia.
  • myeloid disorders include, but are not limited to, acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) (reviewed in Vaickus (1991), Crit Rev. Oncol./Hemotol. 11:267-97 ); lymphoid malignancies include, but are not limited to acute lymphoblastic leukemia (ALL) which includes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM).
  • ALL acute lymphoblastic leukemia
  • CLL chronic lymphocytic leukemia
  • PLL prolymphocytic leukemia
  • HLL hairy cell leukemia
  • WM Waldenstrom's macroglobulinemia
  • malignant lymphomas include, but are not limited to non-Hodgkin lymphoma and variants thereof, peripheral T cell lymphomas, adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease and Reed-Stemberg disease.
  • proliferative breast disease including, e.g ., epithelial hyperplasia, sclerosing adenosis, and small duct papillomas
  • tumors e.g ., stromal tumors such as fibroadenoma, phyllodes tumor, and sarcomas, and epithelial tumors such as large duct papilloma
  • carcinoma of the breast including in situ (noninvasive) carcinoma that includes ductal carcinoma in situ (including Paget's disease) and lobular carcinoma in situ, and invasive (infiltrating) carcinoma including, but not limited to, invasive ductal carcinoma, invasive lobular carcinoma, medullary carcinoma, colloid (mucinous) carcinoma, tubular carcinoma, and invasive papillary carcinoma, and miscellaneous malignant neoplasms.
  • Disorders in the male breast include, but are not limited to, g
  • Examples of cellular proliferative and/or differentiative disorders of the lung include, but are not limited to, bronchogenic carcinoma, including paraneoplastic syndromes, bronchioloalveolar carcinoma, neuroendocrine tumors, such as bronchial carcinoid, miscellaneous tumors, and metastatic tumors; pathologies of the pleura, including inflammatory pleural effusions, noninflammatory pleural effusions, pneumothorax, and pleural tumors, including solitary fibrous tumors (pleural fibroma) and malignant mesothelioma.
  • bronchogenic carcinoma including paraneoplastic syndromes, bronchioloalveolar carcinoma, neuroendocrine tumors, such as bronchial carcinoid, miscellaneous tumors, and metastatic tumors
  • pathologies of the pleura including inflammatory pleural effusions, noninflammatory pleural effusions, pneumothorax, and pleural tumors, including solitary fibrous tumors (pleural fibro
  • Examples of cellular proliferative and/or differentiative disorders of the colon include, but are not limited to, non-neoplastic polyps, adenomas, familial syndromes, colorectal carcinogenesis, colorectal carcinoma, and carcinoid tumors.
  • Examples of cellular proliferative and/or differentiative disorders of the liver include, but are not limited to, nodular hyperplasias, adenomas, and malignant tumors, including primary carcinoma of the liver and metastatic tumors.
  • ovarian tumors such as, tumors of coelomic epithelium, serous tumors, mucinous tumors, endometrioid tumors, clear cell adenocarcinoma, cystadenofibroma, Brenner tumor, surface epithelial tumors; germ cell tumors such as mature (benign) teratomas, monodermal teratomas, immature malignant teratomas, dysgerminoma, endodermal sinus tumor, choriocarcinoma; sex cord-stomal tumors such as, granulosa-theca cell tumors, thecomafibromas, androblastomas, hill cell tumors, and gonadoblastoma; and metastatic tumors such as Krukenberg tumors.
  • ovarian tumors such as, tumors of coelomic epithelium, serous tumors, mucinous tumors, endometrioid tumors, clear cell adenocarcinoma, cystadeno
  • the invention provides methods of treating breast cancer by administering the peptidomimetic macrocycles of the invention.
  • Breast cancer includes invasive breast carcinomas, such as invasive ductal carcinoma, invasive lobular carcinoma, tubular carcinoma, invasive cribriform carcinoma, medullary carcinoma, mucinous carcinoma and other tumours with abundant mucin, cystadenocarcinoma, columnar cell mucinous carcinoma, signet ring cell carcinoma, neuroendocrine tumours (including solid neuroendocrine carcinoma, atypical carcinoid tumour, small cell/oat cell carcinoma, or large cell neuroendocrine carcioma), invasive papillary carcinoma, invasive micropapillary carcinoma, apocrine carcinoma, metaplastic carcinomas, pure epithelial metaplastic carciomas, mixed epithelial/mesenchymal metaplastic carcinomas, lipid-rich carcinoma, secretory carcinoma, oncocytic carcinoma, adenoid cystic carcinoma, acinic cell carcinoma, glycogen-rich clear cell carcinoma,
  • Treatment of breast cancer may be effected in conjunction with any additional therapy, such as a therapy that is part of the standard of care.
  • a surgical technique such as lumpectomy or mastectomy may be performed prior to, during, or following treatment with the peptidomimetic macrocycles of the invention.
  • radiation therapy may be used for the treatment of breast cancer in conjunction with the peptidomimetic macrocycles of the invention.
  • the peptidomimetic macrocycles of the invention are administered in combination with a second therapeutic agent.
  • a second therapeutic agent may be a chemotherapeutic agent such as an individual drug or combination of drugs and therapies.
  • the chemotherapeutic agent can be an adjuvant chemotherapeutic treatment such as CMF (cyclophosphamide, methotrexate, and 5-fluorouracil); FAC or CAF (5-fluorouracil, doxorubicin, cyclophosphamide); AC or CA (doxorubicin and cyclophosphamide); AC-Taxol (AC followed by paclitaxel); TAC (docetaxel, doxorubicin, and cyclophosphamide); FEC (5-fluorouracil, epirubicin and cyclophosphamide); FECD (FEC followed by docetaxel); TC (docetaxel and cyclophosphamide).
  • CMF cyclophosphamide, methotrexate, and 5-fluorouracil
  • FAC or CAF 5-fluorouracil, doxorubicin, cyclophosphamide
  • AC or CA
  • trastuzumab may also be added to the regimen depending on the tumor characteristics (i.e. HER2/neu status) and risk of relapse.
  • Hormonal therapy may also be appropriate before, during or following chemotherapeutic treatment.
  • tamoxifen may be administered or a compound in the category of aromatase inhibitors including, but not limited to aminogluthetimide, anastrozole, exemestane, formestane, letrozole, or vorozole.
  • an antiangiogenic agent may be used in combination therapy for the treatment of breast cancer.
  • the antiangiogenic agent may be an anti-VEGF agent including, but not limited to bevacizumab.
  • the peptidomimetic macrocycles of the invention may be used to treat ovarian cancer.
  • Ovarian cancers include ovarian tumors such as, tumors of coelomic epithelium, serous tumors, mucinous tumors, endometrioid tumors, clear cell adenocarcinoma, cystadenofibroma, Brenner tumor, surface epithelial tumors; germ cell tumors such as mature (benign) teratomas, monodermal teratomas, immature malignant teratomas, dysgerminoma, endodermal sinus tumor, choriocarcinoma; sex cord-stomal tumors such as, granulosa-theca cell tumors, thecomafibromas, androblastomas, hill cell tumors, and gonadoblastoma; and metastatic tumors such as Krukenberg tumors.
  • the peptidomimetic macrocycles of the invention may be administered in conjunction with a second therapy such as a therapy that is part of the standard of care.
  • a second therapy such as a therapy that is part of the standard of care.
  • Surgery, immunotherapy, chemotherapy, hormone therapy, radiation therapy, or a combination thereof are some possible treatments available for ovarian cancer.
  • Some possible surgical procedures include debulking, and a unilateral or bilateral oophorectomy and/or a unilateral or bilateral salpigectomy.
  • Anti-cancer drugs that may be used include cyclophosphamide, etoposide, altretamine, and ifosfamide. Hormone therapy with the drug tamoxifen may be used to shrink ovarian tumors. Radiation therapy may be external beam radiation therapy and/or brachytherapy.
  • the peptidomimetic macrocycles of the invention may be used to treat prostate cancer.
  • Prostate cancers include adenocarcinomas and metastasized adenocarcinomas.
  • the peptidomimetic macrocycles of the invention may be administered in conjunction with a second therapy such as a therapy that is part of the standard of care.
  • Treatment for prostate cancer may involve surgery, radiation therapy, High Intensity Focused Ultrasound (HIFU), chemotherapy, cryosurgery, hormonal therapy, or any combination thereof.
  • Surgery may involve prostatectomy, radical perineal prostatectomy, laparoscopic radical prostatectomy, transurethral resection of the prostate or orchiectomy.
  • Radiation therapy may include external beam radiation therapy and/or brachytherapy.
  • Hormonal therapy may include orchiectomy; administration of antiandrogens such as flutamide, bicalutamide, nilutamide, or cyproterone acetate; medications which inhibit the production of adrenal androgens such as DHEA, such as ketoconazole and aminoglutethimide; and GnRH antagonists or agonists such as Abarelix (Plenaxis®), Cetrorelix (Cetrotide®), Ganirelix (Antagon®), leuprolide, goserelin, triptorelin, or buserelin. Treatment with an anti-androgen agent, which blocks androgen activity in the body, is another available therapy.
  • antiandrogens such as flutamide, bicalutamide, nilutamide, or cyproterone acetate
  • medications which inhibit the production of adrenal androgens such as DHEA, such as ketoconazole and aminoglutethimide
  • GnRH antagonists or agonists such as
  • Such agents include flutamide, bicalutamide, and nilutamide.
  • This therapy is typically combined with LHRH analog administration or an orchiectomy, which is termed a combined androgen blockade (CAB).
  • Chemotherapy includes, but is not limited to, administration of docetaxel, for example with a corticosteroid such as prednisone.
  • Anti-cancer drugs such as doxorubicin, estramustine, etoposide, mitoxantrone, vinblastine, paclitaxel, carboplatin may also be administered to slow the growth of prostate cancer, reduce symptoms and improve the quality of life. Additional compounds such as bisphosphonate drugs may also be administered.
  • the peptidomimetic macrocycles of the invention may be used to treat renal cancer.
  • Renal cancers include, but are not limited to, renal cell carcinomas, metastases from extra-renal primary neoplasms, renal lymphomas, squamous cell carcinomas, juxtaglomerular tumors (reninomas), transitional cell carcinomas, angiomyolipomas, oncocytomas and Wilm's tumors.
  • the peptidomimetic macrocycles of the invention may be administered in conjunction with a second therapy such as a therapy that is part of the standard of care.
  • Treatment for renal cancer may involve surgery, percutaneous therapies, radiation therapies, chemotherapy, vaccines, or other medication.
  • Surgical techniques useful for treatment of renal cancer in combination with the peptidomimetic macrocycles of the invention include nephrectomy, which may include removal of the adrenal gland, retroperitoneal lymph nodes, and any other surrounding tissues affected by the invasion of the tumor.
  • Percutaneous therapies include, for example, image-guided therapies which may involve imaging of a tumor followed by its targeted destruction by radiofrequency ablation or cryotherapy.
  • other chemotherapeutic or other medications useful in treating renal cancer may be alpha-interferon, interleukin-2, bevacizumab, sorafenib, sunitib, temsirolimus or other kinase inhibitors.
  • the invention provides methods of treating pancreatic cancer by administering peptidomimetic macrocycles of the invention, such as a pancreatic cancer selected from the following: an epitheliod carcinoma in the pancreatic duct tissue and an adenocarcinoma in a pancreatic duct.
  • a pancreatic cancer selected from the following: an epitheliod carcinoma in the pancreatic duct tissue and an adenocarcinoma in a pancreatic duct.
  • pancreatic cancer selected from the following: an epitheliod carcinoma in the pancreatic duct tissue and an adenocarcinoma in a pancreatic duct.
  • pancreatic cancer selected from the following: an epitheliod carcinoma in the pancreatic duct tissue and an adenocarcinoma in a pancreatic duct.
  • the most common type of pancreatic cancer is an adenocarcinoma, which occurs in the lining of the pancreatic duct.
  • Radiotherapy may be an option for pancreatic cancer patients, specifically external beam radiation where radiation is focused on the tumor by a machine outside the body. Another option is intraoperative electron beam radiation administered during an operation.
  • Chemotherapy may also be used to treat pancreatic cancer patients.
  • Suitable anti-cancer drugs include, but are not limited to, 5-fluorouracil (5-FU), mitomycin, ifosfamide, doxorubicin, streptozocin, chlorozotocin, and combinations thereof.
  • the methods provided by the invention can provide a beneficial effect for pancreatic cancer patients, by administration of a polypeptide of the invention or a combination of administration of a peptidomimetic macrocycle and surgery, radiation therapy, or chemotherapy.
  • peptidomimetic macrocycles of the invention may be used for the treatment of colon cancer, including but not limited to non-neoplastic polyps, adenomas, familial syndromes, colorectal carcinogenesis, colorectal carcinoma, and carcinoid tumors.
  • Possible treatments available for colon cancer that may be used in conjunction with the peptidomimetic macrocycles of the invention include surgery, chemotherapy, radiation therapy or targeted drug therapy.
  • Radiation therapy may include external beam radiation therapy and/or brachytherapy.
  • Chemotherapy may be used to reduce the likelihood of metastasis developing, shrink tumor size, or slow tumor growth.
  • Chemotherapy is often applied after surgery (adjuvant), before surgery (neo-adjuvant), or as the primary therapy if surgery is not indicated (palliative).
  • exemplary regimens for adjuvant chemotherapy involve the combination of infusional 5-fluorouracil, leucovorin, and oxaliplatin (FOLFOX).
  • First line chemotherapy regimens may involve the combination of infusional 5-fluorouracil, leucovorin, and oxaliplatin (FOLFOX) with a targeted drug such as bevacizumab, cetuximab or panitumumab or infusional 5-fluorouracil, leucovorin, and irinotecan (FOLFIRI) with targeted drug such as bevacizumab, cetuximab or panitumumab.
  • FOLFOX infusional 5-fluorouracil, leucovorin, and oxaliplatin
  • FOLFIRI infusional 5-fluorouracil, leucovorin
  • targeted drug such as bevacizumab, cetuximab or panitumumab.
  • Some embodiments provide methods for the treatment of lung cancer using the peptidomimetic macrocycles of the invention.
  • cellular proliferative and/or differentiative disorders of the lung include, but are not limited to, bronchogenic carcinoma, including paraneoplastic syndromes, bronchioloalveolar carcinoma, neuroendocrine tumors, such as bronchial carcinoid, miscellaneous tumors, and metastatic tumors; pathologies of the pleura, including inflammatory pleural effusions, noninflammatory pleural effusions, pneumothorax, and pleural tumors, including solitary fibrous tumors (pleural fibroma) and malignant mesothelioma.
  • non-small cell lung cancer (NSCLC), which accounts for approximately 80-85% of lung cancers and is divided into squamous cell carcinomas, adenocarcinomas, and large cell undifferentiated carcinomas.
  • Small cell lung cancer e.g. small cell lung carcinomas, accounts for 15-20% of lung cancers.
  • Treatment options for lung cancer include surgery, immunotherapy, radiation therapy, chemotherapy, photodynamic therapy, or a combination thereof.
  • Some possible surgical options for treatment of lung cancer are a segmental or wedge resection, a lobectomy, or a pneumonectomy.
  • Radiation therapy may be external beam radiation therapy or brachytherapy.
  • Some anti-cancer drugs that may be used in chemotherapy to treat lung cancer in combination with the peptidomimetic macrocycles of the invention include cisplatin, carboplatin, paclitaxel, docetaxel, gemcitabine, vinorelbine, irinotecan, etoposide, vinblastine, gefitinib, ifosfamide, methotrexate, or a combination thereof.
  • Photodynamic therapy (PDT) may be used to treat lung cancer patients.
  • the methods described herein can provide a beneficial effect for lung cancer patients, by administration of a peptidomimetic macrocycle or a combination of administration of a peptidomimetic macrocycle and surgery, radiation therapy, chemotherapy, photodynamic therapy, or a combination thereof.
  • Examples of cellular proliferative and/or differentiative disorders of the liver include, but are not limited to, nodular hyperplasias, adenomas, and malignant tumors, including primary carcinoma of the liver and metastatic tumors.
  • Immunoproliferative disorders are disorders of the immune system that are characterized by the abnormal proliferation of the primary cells of the immune system, which includes B cells, T cells and Natural Killer (NK) cells, or by the excessive production of immunoglobulins (also known as antibodies).
  • Such disorders include the general categories of lymphoproliferative disorders, hypergammaglobulinemias, and paraproteinemias. Examples of such disorders include, but are not limited to, X-linked lymphoproliferative disorder, autosomal lymphoproliferative disorder, Hyper-IgM syndrome, heavy chain disease, and cryoglobulinemia.
  • immunoproliferative disorders can be graft versus host disease (GVHD); psoriasis; immune disorders associated with graft transplantation rejection; T cell lymphoma; T cell acute lymphoblastic leukemia; testicular angiocentric T cell lymphoma; benign lymphocytic angiitis; and autoimmune diseases such as lupus erythematosus, Hashimoto's thyroiditis, primary myxedema, Graves' disease, pernicious anemia, autoimmune atrophic gastritis, Addison's disease, insulin dependent diabetes mellitis, good pasture's syndrome, myasthenia gravis, pemphigus, Crohn's disease, sympathetic ophthalmia, autoimmune uveitis, multiple sclerosis, autoimmune hemolytic anemia, idiopathic thrombocytopenia, primary biliary cirrhosis, chronic action hepatitis, ulceratis colitis, Sjogren's syndrome, rheum
  • peptidomimetic macrocycles of the invention may be used for the treatment of cancer in conjunction with alkylating and alkylating-like agents.
  • agents include, for example, nitrogen mustards such as chlorambucil, chlormethine, cyclophosphamide, ifosfamide, and melphalan; nitrosoureas such as carmustine, fotemustine, lomustine, and streptozocin; platinum therapeutic agents such as carboplatin, cisplatin, oxaliplatin, BBR3464, and satraplatin; or other agents, including but not limited to busulfan, dacarbazine, procarbazine, temozolomide, thiotepa, treosulfan, or uramustine.
  • peptidomimetic macrocycles of the invention may be used in conjunction with an antineoplastic agent which is an antimetabolite.
  • an antineoplastic agent may be a folic acid such as aminopterin, methotrexate, pemetrexed, or raltitrexed.
  • the antineoplastic agent may be a purine, including but not limited to cladribine, clofarabine, fludarabine, mercaptopurine, pentostatin, thioguanine.
  • the antineoplastic agent may be a pyrimidine such as capecitabine, cytarabine, fluorouracil, floxuridine, and gemcitabine.
  • peptidomimetic macrocycles of the invention may be used in conjunction with an antineoplastic agent which is an spindle poison/mitotic inhibitor.
  • Agents in this category include taxanes, for example docetaxel and paclitaxel; and vinca alkaloids such as vinblastine, vincristine, vindesine, and vinorelbine.
  • peptidomimetic macrocycles of the invention may be used in combination with an antineoplastic agent which is a cytotoxic/antitumor antibiotic from the anthracycline family such as daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, pixantrone, or valrubicin; an antibiotic from the streptomyces family such as actinomycin, bleomycin, mitomycin, or plicamycin; or hydroxyurea.
  • agents used for combination therapy may be topoisomerase inhibitors including, but not limited to camptothecin, topotecan, irinotecan, etoposide, or teniposide.
  • the antineoplastic agent may be an antibody or antibody-derived agent.
  • a receptor tyrosine kinase-targeted antibody such as cetuximab, panitumumab, or trastuzumab may be used
  • the antibody may be an anti-CD20 antibody such as rituximab or tositumomab, or any other suitable antibody including but not limited to alemtuzumab, bevacizumab, and gemtuzumab.
  • the antineoplastic agent is a photosensitizer such as aminolevulinic acid, methyl aminolevulinate, porfimer sodium, or verteporfm.
  • the antineoplastic agent is a tyrosine kinase inhibitor such as dediranib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, sorafenib, sunitinib, or vandetanib.
  • neoplastic agents suitable in the use of the invention include, for example, alitretinoin, tretinoin, altretamine, amsacrine, anagrelide, arsenic trioxide, asparaginase (pegaspargase), bexarotene, bortezomib, denileukin diftitox, estramustine, ixabepilone, masoprocol, or mitotane.
  • the peptidomimetics macrocycles described herein are used to treat, prevent or diagnose conditions characterized by overactive cell death or cellular death due to physiologic insult, etc.
  • conditions characterized by premature or unwanted cell death are or alternatively unwanted or excessive cellular proliferation include, but are not limited to hypocellular/hypoplastic, acellular/aplastic, or hypercellular/hyperplastic conditions.
  • Some examples include hematologic disorders including but not limited to fanconi anemia, aplastic anemia, thalaessemia, congenital neutropenia, myelodysplasia
  • the peptidomimetics macrocycles of the invention that act to decrease apoptosis are used to treat disorders associated with an undesirable level of cell death.
  • the anti-apoptotic peptidomimetics macrocycles of the invention are used to treat disorders such as those that lead to cell death associated with viral infection, e.g., infection associated with infection with human immunodeficiency virus (HIV).
  • HIV human immunodeficiency virus
  • a wide variety of neurological diseases are characterized by the gradual loss of specific sets of neurons, and the anti-apoptotic peptidomimetics macrocycles of the invention are used, in some embodiments, in the treatment of these disorders.
  • Such disorders include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS) retinitis pigmentosa, spinal muscular atrophy, and various forms of cerebellar degeneration.
  • the cell loss in these diseases does not induce an inflammatory response, and apoptosis appears to be the mechanism of cell death.
  • a number of hematologic diseases are associated with a decreased production of blood cells.
  • These disorders include anemia associated with chronic disease, aplastic anemia, chronic neutropenia, and the myelodysplastic syndromes.
  • disorders of blood cell production such as myelodysplastic syndrome and some forms of aplastic anemia, are associated with increased apoptotic cell death within the bone marrow.
  • disorders could result from the activation of genes that promote apoptosis, acquired deficiencies in stromal cells or hematopoietic survival factors, or the direct effects of toxins and mediators of immune responses.
  • Two common disorders associated with cell death are myocardial infarctions and stroke. In both disorders, cells within the central area of ischemia, which is produced in the event of acute loss of blood flow, appear to die rapidly as a result of necrosis. However, outside the central ischemic zone, cells die over a more protracted time period and morphologically appear to die by apoptosis.
  • the anti-apoptotic peptidomimetics macrocycles of the invention are used to treat all such disorders associated with undesirable cell death.
  • immunologic disorders that are treated with the peptidomimetics macrocycles described herein include but are not limited to organ transplant rejection, arthritis, lupus, IBD, Crohn's disease, asthma, multiple sclerosis, diabetes, etc.
  • neurologic disorders that are treated with the peptidomimetics macrocycles described herein include but are not limited to Alzheimer's Disease, Down's Syndrome, Dutch Type Hereditary Cerebral Hemorrhage Amyloidosis, Reactive Amyloidosis, Familial Amyloid Nephropathy with Urticaria and Deafness, Muckle-Wells Syndrome, Idiopathic Myeloma; Macroglobulinemia-Associated Myeloma, Familial Amyloid Polyneuropathy, Familial Amyloid Cardiomyopathy, Isolated Cardiac Amyloid, Systemic Senile Amyloidosis, Adult Onset Diabetes, Insulinoma, Isolated Atrial Amyloid, Medullary Carcinoma of the Thyroid, Familial Amyloidosis, Hereditary Cerebral Hemorrhage With Amyloidosis, Familial Amyloidotic Polyneuropathy, Scrapie, Creutzfeldt-Jacob Disease, Gerstmann Straussler-Scheinker Syndrome
  • cardiovascular disorders e.g., inflammatory disorders
  • cardiovascular disorders include, but are not limited to, atherosclerosis, myocardial infarction, stroke, thrombosis, aneurism, heart failure, ischemic heart disease, angina pectoris, sudden cardiac death, hypertensive heart disease; non-coronary vessel disease, such as arteriolosclerosis, small vessel disease, nephropathy, hypertriglyceridemia, hypercholesterolemia, hyperlipidemia, xanthomatosis, asthma, hypertension, emphysema and chronic pulmonary disease; or a cardiovascular condition associated with interventional procedures ("procedural vascular trauma"), such as restenosis following angioplasty, placement of a shunt, stent, synthetic or natural excision grafts, indwelling catheter, valve or other implantable devices.
  • Preferred cardiovascular disorders include atherosclerosis, myocardial infarction, aneurism, and stroke.
  • Example 1 Synthesis of Peptidomimetic Macrocycles of the Invention. ⁇ -helical BID and BIM peptidomimetic macrocycles were synthesized, purified and analyzed as previously described ( Walensky et al (2004) Science 305:1466-70 ; Walensky et al (2006) Mol Cell 24:199-210 ) and as indicated below. The macrocycles used in this study are shown below. The corresponding uncrosslinked polypeptides are indicated as "WT Sequence" and represent the natural counterparts of the peptidomimetic macrocycles of the invention.
  • BID-BH3 and BIM-BH3 peptidomimetic macrocycles were designed by replacing two naturally occurring amino acids with the corresponding synthetic amino acids. Substitutions were made at the i and i+4 positions.
  • BID BH3 and BIM-BH3 macrocycles were generated by solid phase peptide synthesis followed by olefin metathesis-based crosslinking of the synthetic amino acids via their olefin-containing side chains. The control sequences for BID and BIM peptidomimetic macrocycles, as well as specific sequence mutations generated are shown above.
  • Nle represents norleucine
  • Aib represents 2-aminoisobutyric acid
  • Chg represents cyclohexylglycine
  • Ac represents acetyl
  • Pr represents propionyl.
  • Amino acids represented as $ connect an all-carbon crosslinker comprising one double bond and wherein each ⁇ -carbon atom to which the crosslinker is attached is additionally substituted with a methyl group.
  • the crosslinker is a linear all-carbon crosslinker comprising eight carbon atoms between the alpha carbons of each amino acid. If a double bond is present, it is positioned between the fourth and fifth carbon atom.
  • the non-natural amino acids were characterized by nuclear magnetic resonance (NMR) spectroscopy (Varian Mercury 400) and mass spectrometry (Micromass LCT). Peptide synthesis was performed either manually or on an automated peptide synthesizer (Applied Biosystems, model 433A), using solid phase conditions, rink amide AM resin (Novabiochem), and Fmoc main-chain protecting group chemistry.
  • Cell lines used in this study are indicated in the table below: Cells Type Source Jurkat human acute T cell leukemia ATCC K562 human chronic myelogeneous leukemia ATCC Karpas299 human T cell lymphoma ATCC MOLT4 human T cell leukemia NCI RPMI8226 human B lymphoblastoma NCI Ramos human B cell lymphoma ATCC Raji human B cell lymphoma ATCC HL-60 Human myeloid leukemia NCI Malme-3M lung malignant melanoma NCI SKMEL2 human malignant melanoma NCI SKMEL5 human malignant melanoma NCI PC3 human prostate adenocarcinoma NCI Caki1 human kidney clear cell carcinoma NCI HCT116 human colorectal carcinoma NCI HT-29 Colorectal adenocarcinoma NCI HEPG2 human hepatocellular carcinoma ATCC MDAMB231 human breast adenocarcinoma NCI MCF7 human breast adenocarcino
  • Cell viability assays shown in Figures 1-32 were performed according to the following protocol. Tumor cell lines were grown in specific serum-supplemented media (growth media) as necessary. A day prior to the initiation of the study, cells were plated at optimal cell density (15,000 to 25,000 cells/well) in 200 ⁇ l growth media in microtiter plates. The next day, cells were washed twice in serum-free/phenol red-free RPMI complete media (assay buffer) and a final volume of 100 ⁇ l assay buffer was added to each well. Human peripheral blood lymphocytes (hPBLs) were isolated from Buffy coats (San Diego Blood Bank) using Ficoll-Paque gradient separation and plated on the day of the experiment at 25,000 cells/well.
  • growth media growth media
  • hPBLs Human peripheral blood lymphocytes
  • Peptidomimetic macrocycles were diluted from 1 mM stocks (100% DMSO) in sterile water to prepare 400 ⁇ M working solutions.
  • the peptidomimetic macrocycles and controls were then diluted 10 or 40 fold or alternatively serially two-fold diluted in assay buffer in dosing plates to provide concentrations of either 40 and 20 ⁇ M or between 1.2 and 40 ⁇ M, respectively.
  • 100 ⁇ L of each dilution was then added to the appropriate wells of the test plate to achieve final concentrations of the peptidomimetic macrocycles equal to 20 or 5 ⁇ M, or between 0.6 to 20 ⁇ M, respectively.
  • Controls included wells without peptidomimetic macrocycles containing the same concentration of DMSO as the wells containing the peptidomimetic macrocycles, wells containing 0.1% Triton X-100, wells containing a chemo cocktail comprised of 1 ⁇ M Velcade, 100 ⁇ M Etoposide and 20 ⁇ M Taxol and wells containing no cells. Plates were incubated for 4 hours at 37°C in humidified 5% CO 2 atmosphere.
  • Figure 1 shows human tumor cell lines treated with SP-1 (20 ⁇ M) and assessed for cell viability by an MTT assay 48 hrs post test article addition. All leukemia/lymphoma lines tested were sensitive to SP-1. In addition, SP-1 also induced apoptosis of several solid tumor lines including three small cell lung carcinoma (SCLC) lines, NCI-H220, NCI-H128 and NCI-H146. Conversely, there were several solid tumor lines resistant to SP-1 including non-small cell lung carcinoma (NSCLC) lines A-549 and H-460, MCF7 (breast cancer) and U251 (glioma).
  • Figure 2 shows seven leukemia/lymphoma human cell lines were treated with 5 ⁇ M of either SP-1 or SP-4 for 48 hrs and assessed for cell viability. As shown, all cell lines exhibited similar sensitivity to both macrocycles at this concentration.
  • Figure 3 shows twelve human solid tumor lines tested for sensitivity to either SP-1 or SP-4 (20 ⁇ M). As shown, there seems to be a cell-specific difference of sensitivity for each macrocycle tested. EC 50 curves for SP-1, SP-2, SP-3, SP-4, SP-5 and SP-6 for individual cell lines are shown in Figures 4-15 .
  • Cell viability assays shown in Figures 34-52 were performed according to a similar protocol. Cells were split at optimal cell density a day prior to the initiation of the study. The next day, cells were washed twice in serum-free Opti-MEM media and 4000 cells/well were added to each well in a final volume of 100 ⁇ l Opti-MEM. For serum-free experiments, macrocycles were diluted from 2 mM stocks (100% DMSO) in sterile water to prepare 400 ⁇ M working solutions. A 40 ⁇ M solution was then generated by ten-fold dilution in assay buffer. The macrocycle and controls were then serially diluted two-fold in assay buffer in dosing plates to provide concentrations between 1.2 and 40 ⁇ M, respectively.
  • macrocycles were diluted from 10 mM stocks (100% DMSO) in sterile water to prepare 1mM working solutions. A 100 ⁇ M solution was generated by ten-fold dilution in assay buffer. The macrocycles and controls were then serially diluted two-fold in assay buffer in dosing plates to provide concentrations between 3 and 100 ⁇ M, respectively. 50 ⁇ L of each dilution was then added to the appropriate wells of the test plate to achieve final concentrations of the macrocycle between 0.6 to 20 ⁇ M (for serum free experiment) or 1.5 to 50 ⁇ M (for 2% serum experiment), respectively.
  • Controls included wells without macrocycles containing the same concentration of DMSO as the macrocycle-containing wells, wells containing 0.1% Triton X-100, and wells containing no cells. Plates were incubated for 24 hours at 37°C in a humidified 5% CO 2 atmosphere. At the end of a 24 hr incubation period, a CellTiter-Glo Luminescent Cell Viability Assay was performed according to manufacturer's instructions (Promega, catalog #G7571) and the luminescence was measured using a BIO-TEK synergy HT Plate reader. Values were plotted as percent viable, i.e. as a percentage of the negative control value (derived from cells exposed to DMSO only). All assays were performed in duplicate.
  • Example 4 Efficacy of peptidomimetic macrocycles in a human leukemia xenograft model.
  • SEMK2-LN cells stably expressing luciferase were generated as previously described ( Armstrong et al (2003) Cancer Cell 3:173-83 ). 6-8 week old female NOD-SCID mice (Jackson Laboratory) were injected with 5 x 10 6 SEMK2-LN cells by tail vein. The animals were imaged as described ( Walensky et al., Science 305:1466-1470 (2004 )) using Xenogen's In Vivo Imaging System (Caliper Life Sciences) and total body bioluminescence quantified by integration of photonic flux (photons/sec) (Living Imaging Software for Xenogen In Vivo Imaging System, Caliper Life Sciences).
  • mice were imaged on days 8 and 12, post-injection of leukemic cells, to identify animals with established leukemia. On day 12, prior to the initiation of treatment (treatment day 1), animals were divided into cohorts with statistically equivalent bioluminescence. Leukemic mice received a daily tail vein injection of peptidomimetic macrocycle at 3 or 10 mg/Kg/day for 21 days or 30 mg/Kg/day for 12 days. Animals were imaged at days 1, 3, 5, 7, 9, 13 and 17 during treatment, and the resulting tumor reduction is shown in Figure 18 .
  • mice 6-8 week old Balb/c or KM female mice were immunized with unconjugated SP-1 or SP-4.
  • Sera were collected pre-immunization and 25 ⁇ g of each peptide in D5W were injected by tail vein using the following immunization schedule: sera were collected seven days following the first two immunizations at days 1 and 14 and 14 days following the third and fourth immunizations.
  • Antibody titers were determined by indirect ELISA. In brief, microtiter plates were coated with either SP-1 or SP-3 (5 ⁇ g/ml) overnight at 4°C.
  • the plates were washed 5 times with PBS/0.05%Tween20 (PBST) and blocked with 5% non-fat milk/PBST for one hour at 37°C, followed by additional washing with PBST.
  • the anti-sera was serially diluted and added to the coated plates for 1 hr at 37°C.
  • the plates were then washed extensively with PBST and further incubated with either HRP-conjugated anti-mouse IgG or IgM for 1 hr at 37°C and washed 5 times with PBST prior to the addition of HRP substrate. Plates were incubated at room temperature for 10 minutes and the reaction stopped with 0.5 M oxalic acid solution.
  • Lyophilized SP-1 was dissolved in ddH 2 O to a final concentration of 50 ⁇ M. Tm was determined by measuring the circular dichroism (CD) spectra in a Jasco-810 spectropolarimeter at a fixed wavelength of 222 nm between the temperatures of 5-95°C. The following parameters were used for the measurement: data pitch, 0.1°C; bandwidth, 1nm and path length, 0.1cm averaging the signal for 16 seconds. The results are shown in Figure 21 .
  • CD circular dichroism
  • the IV dose formulation was prepared by dissolving SP-1 or SP-4 in 5 % DMSO/ D5W to achieve a 10 mg/Kg/dose.
  • Canulated Crl:CD ® SD male rats (7-8 weeks old, Charles River Laboratories) were used in these studies. Intravenous doses were administered via the femoral cannula and the animals were dosed at 10 mL/kg per single injection. Blood for pharmacokinetic analysis was collected at 10 time points (0.0833, 0.25, 0.5,1, 2, 4, 6, 8, 12 and 24 hrs post-dose). Animals were terminated (without necropsy) following their final sample collection.
  • Plasma samples were centrifuged ( ⁇ 1500 x g) for 10 min at ⁇ 4 °C. Plasma was prepared and transferred within 30 min of blood collection/centrifugation to fresh tubes that were frozen and stored in the dark at ⁇ -70 °C until they were prepared for LC-MS/MS analysis.
  • Sample extraction was achieved by adding 10 ⁇ L of 50% formic acid to 100 ⁇ L plasma (samples or standards), following by vortexing for 10 seconds. 500 ⁇ L acetonitrile was added to the followed by vortexing for 2 minutes and centrifuged at 14,000rpm for 10 minutes at ⁇ 4°C. Supernatants were transferred to clean tubes and evaporated on trubovap ⁇ 10 psi at 37°C. Prior to LC-MS/MS analysis samples were reconstituted with 100 ⁇ L of 50:50 acetonitrile:water.
  • LC-MS/MS The following LC-MS/MS method was used.
  • the LC-MS/MS instruments used was an API 365 (Applied Biosystems).
  • the analytical column was a Phenomenex Synergi (4 ⁇ , Polar-RP, 50mm x 2 mm) and mobile phases A (0.1 % formic acid in water) and B (0.1 % formic acid in methanol) were pumped at a flow rate of 0.4 ml/min to achieve the following gradient: Time (min) %B 0 15 0.5 15 1.5 95 4.5 95 4.6 15 8.0 Stop MRM: 814.0 to 374.2 (positive ionization)
  • Cells e.g. Jurkat cells
  • RPMI1640 medium with 2mM L-Glutamine (Invitrogen) and supplemented with 10% FBS and 1% penicillin-Streptomycin.
  • Cells were subcultured a day prior to the day of experiment to keep them in an exponential growing phase.
  • FACS Fluorescence-Activated Cell Sorting
  • Test compounds were diluted to 2mM stock in DMSO, followed by dilution to 400 ⁇ M in sterile water; further dilution to 100 ⁇ M was done using OptiMEM. Thus 100 ⁇ l of 100uM FITC-labeled peptidomimetic macrocycle was then added to appropriate wells to achieve a final concentration of 10 ⁇ M in 1ml volume. Plates were returned to 37°C, 5% CO 2 incubators for designated time points. At the end of each time point, the cell suspension was diluted with media, washed twice and subjected to trypsin (0.25%) for 15 min at 37°C. Cells were then washed with OptiMem and finally resuspended in 500 ⁇ l of PBS. Cellular fluorescence was measured using Beckman Coulter FACS instrument counting at least 20000 events per sample. Analysis was done using Summit version 4, Dako Colorado, Inc .
  • Protein-ligand binding experiments were conducted according to the following representative procedure outlined for a system-wide control experiment using 1 ⁇ M SP-4 plus 5 ⁇ M Bcl-x L .
  • a 1 ⁇ L DMSO aliquot of a 40 ⁇ M stock solution of peptidomimetic macrocycle was dissolved in 19 ⁇ L of PBS (Phosphate-buffered saline: 50 mM, pH 7.5 Phosphate buffer containing 150 mM NaCl).
  • PBS Phosphate-buffered saline: 50 mM, pH 7.5 Phosphate buffer containing 150 mM NaCl.
  • the resulting solution was mixed by repeated pipetting and clarified by centrifugation at 10 000g for 10 min.
  • To a 4 ⁇ L aliquot of the resulting supernatant was added 4 ⁇ L of 10 ⁇ M BCL-x L in PBS.
  • Each 8.0 ⁇ L experimental sample thus contained 40 pmol (1.5 ⁇ g) of protein at 5.0 ⁇ M concentration in PBS plus 1 ⁇ M peptidomimetic macrocycle and 2.5% DMSO.
  • Duplicate samples thus prepared for each concentration point were incubated for 60 min at room temperature, and then chilled to 4 °C prior to size-exclusion chromatography-LC-MS analysis of 5.0 ⁇ L injections.
  • Samples containing a target protein, protein-ligand complexes, and unbound compounds were injected onto an SEC column, where the complexes were separated from non-binding component by a rapid SEC step.
  • the SEC column eluate was monitored using UV detectors to confirm that the early-eluting protein fraction, which elutes in the void volume of the SEC column, was well resolved from unbound components that are retained on the column.
  • the peak containing the protein and protein-ligand complexes elutes from the primary UV detector, it entered a sample loop where it was excised from the flow stream of the SEC stage and transferred directly to the LC-MS via a valving mechanism.
  • the (M + 3H) 3+ ion of ALRN-0034 is observed by ESI-MS at m/z 883.8, confirming the detection of the protein-ligand complex.
  • a mixture of ligands at 40 ⁇ M per component was prepared by combining 2 ⁇ L aliquots of 400 ⁇ M stocks of each of the three compounds with 14 ⁇ L of DMSO. Then, 1 ⁇ L aliquots of this 40 ⁇ M per component mixture were combined with 1 ⁇ L DMSO aliquots of a serially diluted stock solution of titrant peptidomimetic macrocycle (10, 5, 2.5,..., 0.078 mM). These 2 ⁇ L samples were dissolved in 38 ⁇ L of PBS. The resulting solutions were mixed by repeated pipetting and clarified by centrifugation at 10 000g for 10 min.
  • each 8.0 ⁇ L experimental sample thus contained 40 pmol (1.5 ⁇ g) of protein at 5.0 ⁇ M concentration in PBS plus 0.5 ⁇ M ligand, 2.5% DMSO, and varying concentrations (125, 62.5, ..., 0.98 ⁇ M) of the titrant peptidomimetic macrocycle.
  • Duplicate samples thus prepared for each concentration point were incubated at room temperature for 60 min, then chilled to 4 °C prior to SEC-LC-MS analysis of 2.0 ⁇ L injections.
  • Cells e.g. INA-6 or Jurkat cells
  • INA-6 cells were cultured in suspension in RPMI1640 medium with 2mM L-Glutamine and(Invitrogen) supplemented with 10% FBS and 1% penicillin-Streptomycin as well as 1 ng/ml of recombinant human IL-6 supplements in the case of INA-6 cells.
  • Cells were subcultured a day prior to the day of experiment to keep them in an exponential growing phase.
  • exponentially growing cells were seeded in 0.9ml of serum-free medium at density of 0.5 X 10 6 cells. Cells were allowed to settle down until compounds were diluted.
  • Test compounds were diluted to 2mM stock in DMSO, followed by dilution to 400 ⁇ M in sterile water; further dilution to 100 ⁇ M was done using OptiMEM. Thus 100 ⁇ l of 100uM FITC-labeled peptidomimetic macrocycle was then added to appropriate wells to achieve a final concentration of 10 ⁇ M in 1ml volume. Plates were returned to 37°C, 5% CO 2 incubators for designated time points. If needed, further dilutions of test compounds were also prepared in OptiMEM. At the end of each time point, cells were harvested, washed twice with RPMI supplemented with FBS, and washed once with PBS + 0.5% BSA.
  • Pelleted cells were resuspended and incubated with 0.25% Trypsin-EDTA for 15 min at 37°C, 5%CO 2 . Post-incubation, cells were washed with media containing serum once and twice with PBS with 0.5% BSA. At the end of washes, cells were lysed with Triton X-100-containing cell lysis buffer from Cell Signaling Technologies. Fluorescence intensity was measured on a BioTek Synergy 4 instrument. Dilutions of FITC-labeled peptidomimetic macrocycles for the standard curves made in cell lysis buffer and were used for quantitation of the amount of peptidomimetic macrocycles in cells. Analysis was done using Gen 5 software provided by Biotek Inc.
  • Example 13 Efficacy of peptidomimetic macrocycles in an orthotopic prostate tumor model.
  • mice were used 7-10 weeks old. Male nu/nu mice were anesthetized and incisions along the posterior midline of their abdomens, right above the prostate, were created. The bladder was retracted and pressed lightly to expose the prostate. PC-3M-luc-C6 cells (5x10 5 ) were slowly injected into either dorsal prostatic lobe. The peritoneal incision was sutured and the skin was closed. The mice were given buprenorphine (0.1mg/kg in 50 ⁇ l) subcutaneously after the surgical procedure.
  • PC-3M-luc-C6 cells 5x10 5
  • mice were first imaged on day 7 after wound healing. The final 50 mice were grouped into 5 groups (10 mice per group) based on BLI before the start of the treatment. The experimental mice were imaged twice weekly starting from day 14 for 2.5 weeks. At the end of the experiment, the tumors were dissected and weighed. The tumors were then cut into two pieces for snap freeze and fixed in 10% formalin. Test compound treatment was initiated after two stable or increasing bioluminescent signals were registered from the tumor cell inoculation site. Several test groups were used: Group 1 (Vehicle, IV daily dosing), Group 2 (test compound, IV daily dosing at 10mg/kg), Group 3 (Vehicle, i.p. daily dosing), Group 4 (test compound, i.p.
  • test peptidomimetic macrocycles were formulated as follows. Only low retention/siliconized plastic tubes and tips were used. A 60 mg/mL stock solution of each peptidomimetic macrocycle was prepared by dissolving 140 mg of each macrocycle into 2.3 mL of 100% DMSO. The stock solution was divided into 10 aliquots of 0.23 mL for daily dosing (14 mg per vial) and kep frozen at -20C. One vial was thawed on each day of dosing.
  • Example 14 Orthotopic xenograft tumor model using ovarian cancer (SKOV3-Luc) tumors.
  • Efficacy is determined by comparison of the tumor burden between peptidomimetic macrocycle and vehicle control treated animals.
  • Tumor growth/volume is monitored by BLI after IP injection of 150 mg/kg D-luciferin and imaged by IVIS Imaging System both dorsally and ventrally. Metastatic lesions can be imaged by shielding the primary tumor bioluminescence.
  • the animals are humanely euthanized and the ovarian tumor is excised, weighed and prepared for subsequent analysis.
  • Example 15 Orthotopic xenograft tumor model using breast cancer (MDA-MB-231-Luc) tumors.
  • Example 16 Subcutaneous xenograft tumor model using melanoma (A375) or small cell lung cancer (NCI-H-82) tumors.
  • An optimized amount of tumor cells is injected into the flank of anesthetized NOD/SCID or nu/nu mice, as required, by subcutaneous injection.
  • the animals are sorted into control and treatment groups (10 mice/group). Animals are treated by daily injection (IP, IV or SC) of test compound (low, medium, and high doses) and vehicle control for 10, 14 and/or 21 days, as needed. Efficacy is determined by comparison of the tumor volume between test compound and vehicle control treated animals. Tumor growth/volume is monitored by external caliper measurement (LxWxD). At the conclusion of the experiment, the animals are humanely euthanized and the tumor is excised, weighed and prepared for subsequent analysis.
  • Example 17 Metastatic tumor model using metastatic breast cancer (MDA-MB-231-Met-Luc) tumors.
  • Anesthetized NOD/SCID mice (9 weeks old, female) are injected with the optimized amount of MDA-MB-213-MET-Luc cells stably expressing firefly luciferase into the left ventricle of the heart (hence directly into arterial system).
  • a successful intracardiac injection is indicated by day zero images showing a systemic bioluminescence distributed throughout the animals and only mice with evidence of a satisfactory injection will remain in the experiment.
  • the animals are sorted into control and treatment groups (10 mice/group). Animals are treated by daily injection (IP, IV or SC) of test compound (low, medium, and high doses) and vehicle control for 10, 14 and/or 21 days, as needed.

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Families Citing this family (52)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2669696A1 (fr) 2006-11-15 2008-05-22 Dana-Farber Cancer Institute, Inc. Peptides maml stabilises et leurs utilisations
US7981998B2 (en) * 2006-12-14 2011-07-19 Aileron Therapeutics, Inc. Bis-sulfhydryl macrocyclization systems
CN105061577A (zh) * 2006-12-14 2015-11-18 爱勒让治疗公司 双巯基大环化系统
JP5649825B2 (ja) 2007-01-31 2015-01-07 デイナ ファーバー キャンサー インスティチュート，インコーポレイテッド 安定化させたｐ５３ペプチドおよびその使用法
BRPI0807578A2 (pt) 2007-02-23 2021-06-15 Aileron Therapeutics, Inc. macrociclo peptidomimético, composto, kit e métodos para sintetizar um macrociclo peptidomimético
KR101525754B1 (ko) 2007-03-28 2015-06-09 프레지던트 앤드 펠로우즈 오브 하바드 칼리지 스티칭된 폴리펩티드
CA2714251C (fr) 2008-02-08 2017-08-15 Aileron Therapeutics, Inc. Macrocycles peptidomimetiques therapeutiques
EP2342222B1 (fr) * 2008-09-22 2018-03-21 Aileron Therapeutics, Inc. Macrocycles peptidomimétiques
JP2012509902A (ja) 2008-11-24 2012-04-26 エルロン・セラピューティクス・インコーポレイテッド 改善された特性を有するペプチド模倣大環状分子
JP2012515172A (ja) 2009-01-14 2012-07-05 エルロン・セラピューティクス・インコーポレイテッド ペプチド模倣大環状分子
US20130072439A1 (en) * 2009-09-22 2013-03-21 Huw M. Nash Peptidomimetic macrocycles
AU2010306718A1 (en) * 2009-10-14 2012-05-24 Aileron Therapeutics, Inc. Improved peptidomimetic macrocycles
GB0918579D0 (en) 2009-10-22 2009-12-09 Imp Innovations Ltd Gadd45beta targeting agents
EP2575453B1 (fr) 2010-05-28 2018-08-01 The Board of Regents of the University of Texas System Composés oligo-benzamide et leur utilisation
CN108570097A (zh) 2010-08-13 2018-09-25 爱勒让治疗公司 拟肽大环化合物
WO2012051405A1 (fr) 2010-10-13 2012-04-19 Bristol-Myers Squibb Company Procédés de préparation de macrocycles et peptides stabilisés par des macrocycles
WO2012065181A2 (fr) 2010-11-12 2012-05-18 Dana Farber Cancer Institute, Inc. Thérapies et diagnostics du cancer
WO2012083078A2 (fr) 2010-12-15 2012-06-21 The Research Foundation Of State University Of New York Peptides et protéines réticulés, leurs procédés de fabrication et leurs utilisations
MX358886B (es) 2011-10-18 2018-08-31 Aileron Therapeutics Inc Macrociclos peptidomimeticos.
WO2013078288A1 (fr) * 2011-11-23 2013-05-30 The Board Of Regents Of The University Of Texas System Composés d'oligo-benzamides pour une utilisation dans le traitement de cancers
WO2013078277A1 (fr) 2011-11-23 2013-05-30 The Board Of Regents Of The University Of Texas System Composés d'oligo-benzamides et leur utilisation dans le traitement de cancers
US9464125B2 (en) * 2012-02-03 2016-10-11 The Trustees Of Princeton University Engineered potent cytotoxic stapled BH3 peptides
US8987414B2 (en) 2012-02-15 2015-03-24 Aileron Therapeutics, Inc. Triazole-crosslinked and thioether-crosslinked peptidomimetic macrocycles
SG11201404648PA (en) 2012-02-15 2014-09-26 Aileron Therapeutics Inc Peptidomimetic macrocycles
WO2014004376A2 (fr) * 2012-06-26 2014-01-03 Del Mar Pharmaceuticals Méthodes de traitement de malignités résistantes à un inhibiteur de tyrosine kinase chez des patients ayant des polymorphismes génétiques ou des dérégulations ou des mutations d'ahi1 à l'aide de dianhydrogalactitol, diacétyldianhydrogalactitol, dibromodulcitol ou des analogues ou dérivés correspondants
EP2914256B1 (fr) 2012-11-01 2019-07-31 Aileron Therapeutics, Inc. Acides aminés disubstitués et procédés de préparation et d'utilisation de ceux-ci
WO2014138429A2 (fr) 2013-03-06 2014-09-12 Aileron Therapeutics, Inc. Macrocycles peptidomimétiques et leur utilisation dans la régulation de hif1alpha
WO2014168986A1 (fr) 2013-04-08 2014-10-16 Brown Dennis M Bénéfice thérapeutique de composés chimiques administrés de façon sous-optimale
ES2927607T3 (es) 2013-09-13 2022-11-08 Scripps Research Inst Agentes terapéuticos modificados y composiciones de los mismos
US10039809B2 (en) 2013-12-18 2018-08-07 The California Institute For Biomedical Research Modified therapeutic agents, stapled peptide lipid conjugates, and compositions thereof
EP3126375B1 (fr) * 2014-04-02 2023-12-13 University of Rochester Substances peptimomimétiques macrocycliques pour mimétisme d'hélice alpha
EP3125920B1 (fr) 2014-04-04 2020-12-23 Del Mar Pharmaceuticals Dianhydrogalactitol, diacetyldianhydrogalactitol ou dibromodulcitol dans le traitement du carcinome non à petites cellules des poumons et du cancer des ovaires
CN112972378A (zh) * 2014-09-24 2021-06-18 艾瑞朗医疗公司 拟肽大环化合物及其制剂
JP2017533889A (ja) 2014-09-24 2017-11-16 エルロン・セラピューティクス・インコーポレイテッドＡｉｌｅｒｏｎ Ｔｈｅｒａｐｅｕｔｉｃｓ，Ｉｎｃ． ペプチド模倣大環状分子およびその使用
CA2979999A1 (fr) * 2015-03-18 2016-09-22 Massachusetts Institute Of Technology Peptides de liaison a mcl-1 selectifs
EP3294318A4 (fr) 2015-03-20 2019-04-03 Aileron Therapeutics, Inc. Macrocycles peptidomimétiques et leurs utilisations
US10059741B2 (en) 2015-07-01 2018-08-28 Aileron Therapeutics, Inc. Peptidomimetic macrocycles
US10023613B2 (en) 2015-09-10 2018-07-17 Aileron Therapeutics, Inc. Peptidomimetic macrocycles as modulators of MCL-1
CA2998528A1 (fr) 2015-09-22 2017-03-30 The Regents Of The University Of California Cytotoxines modifiees et leur utilisation therapeutiques
US10286079B2 (en) 2015-09-22 2019-05-14 The Regents Of The University Of California Modified cytotoxins and their therapeutic use
EP3368544B1 (fr) 2015-10-27 2020-06-17 H. Hoffnabb-La Roche Ag Peptides macrocycliques contre acetinobacter baumannii
US20190077840A1 (en) * 2015-10-30 2019-03-14 Massachusetts Institute Of Technology Selective mcl-1 binding peptides
US11021514B2 (en) 2016-06-01 2021-06-01 Athira Pharma, Inc. Compounds
US11198715B2 (en) 2016-07-22 2021-12-14 Massachusetts Institute Of Technology Selective Bfl-1 peptides
EP3388444A1 (fr) 2017-04-10 2018-10-17 F. Hoffmann-La Roche AG Macrocycles peptidiques antibactériens et utilisation associée
US11524979B2 (en) 2017-06-15 2022-12-13 University Of Washington Macrocyclic polypeptides
US11505573B2 (en) 2018-03-28 2022-11-22 Hoffmann-La Roche Inc. Peptide macrocycles against Acinetobacter baumannii
US11819532B2 (en) 2018-04-23 2023-11-21 Hoffmann-La Roche Inc. Peptide macrocycles against Acinetobacter baumannii
US11091522B2 (en) 2018-07-23 2021-08-17 Aileron Therapeutics, Inc. Peptidomimetic macrocycles and uses thereof
WO2020060975A1 (fr) 2018-09-17 2020-03-26 Massachusetts Institute Of Technology Peptides sélectifs pour des protéines de la famille bcl-2
US20200289609A1 (en) * 2019-03-15 2020-09-17 Aileron Therapeutics, Inc. Peptidomimetic macrocycles and uses thereof
WO2023069332A1 (fr) * 2021-10-22 2023-04-27 Merck Sharp & Dohme Llc Peptides macrocycliques perméables aux cellules utiles pour l'inhibition du site de liaison à la coiffe d'eif4e

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5364851A (en) 1991-06-14 1994-11-15 International Synthecon, Llc Conformationally restricted biologically active peptides, methods for their production and uses thereof
US5446128A (en) 1993-06-18 1995-08-29 The Board Of Trustees Of The University Of Illinois Alpha-helix mimetics and methods relating thereto
US5811515A (en) 1995-06-12 1998-09-22 California Institute Of Technology Synthesis of conformationally restricted amino acids, peptides, and peptidomimetics by catalytic ring closing metathesis
US5824483A (en) 1994-05-18 1998-10-20 Pence Inc. Conformationally-restricted combinatiorial library composition and method
US6713280B1 (en) 1999-04-07 2004-03-30 Thomas Jefferson University Enhancement of peptide cellular uptake
US20050250680A1 (en) 2003-11-05 2005-11-10 Walensky Loren D Stabilized alpha helical peptides and uses thereof
US7192713B1 (en) 1999-05-18 2007-03-20 President And Fellows Of Harvard College Stabilized compounds having secondary structure motifs
US7202332B2 (en) 2004-05-27 2007-04-10 New York University Methods for preparing internally constrained peptides and peptidomimetics

Family Cites Families (56)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US753819A (en) * 1903-04-08 1904-03-01 George Jones Atkins Electrode.
US5712418A (en) * 1989-10-23 1998-01-27 Research Corporation Technologies, Inc. Synthesis and use of amino acid fluorides as peptide coupling reagents
DE69118826T2 (de) 1990-11-27 1996-11-14 Fuji Photo Film Co Ltd Propenamidderivate, deren Polymere, Copolymere und deren Verwendung
CA2196061C (fr) * 1992-04-03 2000-06-13 Robert H. Grubbs Complexes carbeniques de ruthenium et d'osmium a haute activite pour reactions de metathese des olefines, et leur procede de synthese
US5411860A (en) * 1992-04-07 1995-05-02 The Johns Hopkins University Amplification of human MDM2 gene in human tumors
US5622852A (en) * 1994-10-31 1997-04-22 Washington University Bcl-x/Bcl-2 associated cell death regulator
IL109943A (en) * 1994-06-08 2006-08-01 Develogen Israel Ltd Conformationally constrained backbone cyclized peptide analogs
US6407059B1 (en) * 1994-06-08 2002-06-18 Peptor Limited Conformationally constrained backbone cyclized peptide analogs
US5807746A (en) 1994-06-13 1998-09-15 Vanderbilt University Method for importing biologically active molecules into cells
US7553929B2 (en) * 1994-06-13 2009-06-30 Vanderbilt University Cell permeable peptides for inhibition of inflammatory reactions and methods of use
US5770377A (en) * 1994-07-20 1998-06-23 University Of Dundee Interruption of binding of MDM2 and P53 protein and therapeutic application thereof
US5672584A (en) * 1995-04-25 1997-09-30 The University Of Kansas Cyclic prodrugs of peptides and peptide nucleic acids having improved metabolic stability and cell membrane permeability
AU4385696A (en) * 1996-01-18 1997-08-11 Christian Gronhoj Larsen Synthetic il-10 analogues
US5663316A (en) * 1996-06-18 1997-09-02 Clontech Laboratories, Inc. BBC6 gene for regulation of cell death
US7083983B2 (en) * 1996-07-05 2006-08-01 Cancer Research Campaign Technology Limited Inhibitors of the interaction between P53 and MDM2
US5955593A (en) * 1996-09-09 1999-09-21 Washington University BH3 interacting domain death agonist
US5965703A (en) * 1996-09-20 1999-10-12 Idun Pharmaceuticals Human bad polypeptides, encoding nucleic acids and methods of use
US5856445A (en) * 1996-10-18 1999-01-05 Washington University Serine substituted mutants of BCL-XL /BCL-2 associated cell death regulator
US6271198B1 (en) * 1996-11-06 2001-08-07 Genentech, Inc. Constrained helical peptides and methods of making same
EP1015578A4 (fr) * 1997-09-17 2004-12-01 Walker And Eliza Hall Inst Of Molecules therapeutiques
US6165732A (en) 1997-10-14 2000-12-26 Washington University Method for identifying apoptosis modulating compounds
US6030997A (en) * 1998-01-21 2000-02-29 Eilat; Eran Acid labile prodrugs
PL347103A1 (en) 1998-04-15 2002-03-25 Aventis Pharm Prod Inc Process for the preparation of resin-bound cyclic peptides
US6326354B1 (en) * 1998-08-19 2001-12-04 Washington University Modulation of apoptosis with bid
IL145406A0 (en) * 1999-03-29 2002-06-30 Procter & Gamble Melanocortin receptor ligands
EP1210098A1 (fr) * 1999-04-07 2002-06-05 Thomas Jefferson University Amelioration de la fixation cellulaire
US6495674B1 (en) 2000-02-25 2002-12-17 The Salk Institute For Biological Studies Evectins and their use
US6703382B2 (en) * 2000-08-16 2004-03-09 Georgetown University Medical Center Small molecule inhibitors targeted at Bcl-2
EP1343884A2 (fr) 2000-12-19 2003-09-17 The Johns Hopkins University Proteine jfy induisant l'apoptose rapide
US6815426B2 (en) * 2001-02-16 2004-11-09 E. I. Du Pont De Nemours And Company Angiogenesis-inhibitory tripeptides, compositions and their methods of use
EP1469871A4 (fr) * 2001-12-31 2006-08-23 Dana Farber Cancer Inst Inc Methode de traitement de l'apoptose et compositions associees
SE0201863D0 (en) 2002-06-18 2002-06-18 Cepep Ab Cell penetrating peptides
WO2004022580A2 (fr) * 2002-09-09 2004-03-18 Dana-Farber Cancer Institute, Inc. Peptides bh3 et leur methode d'utilisation
US7166575B2 (en) * 2002-12-17 2007-01-23 Nastech Pharmaceutical Company Inc. Compositions and methods for enhanced mucosal delivery of peptide YY and methods for treating and preventing obesity
US20070032417A1 (en) 2002-12-24 2007-02-08 Walter And Eliza Hall Institute Of Medical Research Peptides and therapeutic uses thereof
AU2004204942A1 (en) * 2003-01-08 2004-07-29 Xencor, Inc Novel proteins with altered immunogenicity
JP2007537989A (ja) 2003-10-16 2007-12-27 アプラーゲン ゲゼルシャフト ミット ベシュレンクテル ハフツング 安定化ペプチド
WO2005090388A1 (fr) 2004-03-19 2005-09-29 The University Of Queensland Alpha-mimetiques helicoidales, leurs utilisations et leurs procedes de production
US7501397B2 (en) 2004-06-04 2009-03-10 The Brigham And Women's Hospital, Inc. Helical peptidomimetics with enhanced activity
US7842815B2 (en) * 2004-06-17 2010-11-30 Infinity Pharmaceuticals, Inc. Compounds and methods for inhibiting the interaction of BCL proteins with binding partners
CN101006065B (zh) * 2004-06-17 2012-07-11 英菲尼蒂发现公司 抑制bcl蛋白与结合伴侣的相互作用的化合物和方法
WO2006103666A2 (fr) 2005-03-28 2006-10-05 Yeda Research And Development Co. Ltd. Polypeptides bid isoles, polynucleotides les codant et anticorps diriges contre ces polypeptides, methodes d'utilisation pour induire l'arret du cycle cellulaire ou l'apoptose
US7745573B2 (en) 2006-02-17 2010-06-29 Polychip Pharmaceuticals Pty Ltd. Conotoxin analogues and methods for synthesis of intramolecular dicarba bridge-containing peptides
US7538190B2 (en) 2006-02-17 2009-05-26 Polychip Pharmaceuticals Pty Ltd Methods for the synthesis of two or more dicarba bridges in organic compounds
US7981998B2 (en) * 2006-12-14 2011-07-19 Aileron Therapeutics, Inc. Bis-sulfhydryl macrocyclization systems
CN105061577A (zh) * 2006-12-14 2015-11-18 爱勒让治疗公司 双巯基大环化系统
BRPI0807578A2 (pt) * 2007-02-23 2021-06-15 Aileron Therapeutics, Inc. macrociclo peptidomimético, composto, kit e métodos para sintetizar um macrociclo peptidomimético
KR101525754B1 (ko) * 2007-03-28 2015-06-09 프레지던트 앤드 펠로우즈 오브 하바드 칼리지 스티칭된 폴리펩티드
US10716828B2 (en) * 2007-05-02 2020-07-21 Dana-Farber Cancer Institute, Inc. Methods of modulating cellular homeostatic pathways and cellular survival
WO2009042237A2 (fr) * 2007-09-26 2009-04-02 Dana Farber Cancer Institute Procédés et compositions pour moduler des polypeptides de la famille bcl-2
US20100295201A1 (en) * 2007-11-20 2010-11-25 Asahi Kasei Construction Materials Corporation Process for producing heat curing resin foamed plate
CA2714251C (fr) 2008-02-08 2017-08-15 Aileron Therapeutics, Inc. Macrocycles peptidomimetiques therapeutiques
EP2310407A4 (fr) 2008-04-08 2011-09-14 Aileron Therapeutics Inc Macrocycles peptidomimétiques biologiquement actifs
EP2342222B1 (fr) * 2008-09-22 2018-03-21 Aileron Therapeutics, Inc. Macrocycles peptidomimétiques
JP2012509902A (ja) 2008-11-24 2012-04-26 エルロン・セラピューティクス・インコーポレイテッド 改善された特性を有するペプチド模倣大環状分子
AU2010306718A1 (en) * 2009-10-14 2012-05-24 Aileron Therapeutics, Inc. Improved peptidomimetic macrocycles

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5364851A (en) 1991-06-14 1994-11-15 International Synthecon, Llc Conformationally restricted biologically active peptides, methods for their production and uses thereof
US5446128A (en) 1993-06-18 1995-08-29 The Board Of Trustees Of The University Of Illinois Alpha-helix mimetics and methods relating thereto
US5824483A (en) 1994-05-18 1998-10-20 Pence Inc. Conformationally-restricted combinatiorial library composition and method
US5811515A (en) 1995-06-12 1998-09-22 California Institute Of Technology Synthesis of conformationally restricted amino acids, peptides, and peptidomimetics by catalytic ring closing metathesis
US6713280B1 (en) 1999-04-07 2004-03-30 Thomas Jefferson University Enhancement of peptide cellular uptake
US7192713B1 (en) 1999-05-18 2007-03-20 President And Fellows Of Harvard College Stabilized compounds having secondary structure motifs
US20050250680A1 (en) 2003-11-05 2005-11-10 Walensky Loren D Stabilized alpha helical peptides and uses thereof
US7202332B2 (en) 2004-05-27 2007-04-10 New York University Methods for preparing internally constrained peptides and peptidomimetics

Non-Patent Citations (50)

* Cited by examiner, † Cited by third party
Title
"Bioorganic Chemistry: Peptides and Proteins", 1998, OXFORD UNIVERSITY PRESS
ANNIS, D. A.; NAZEF, N.; CHUANG, C. C.; SCOTT, M. P.; NASH, H. M.: "A General Technique to Rank Protein-Ligand Binding Affinities and Determine Allosteric vs. Direct Binding Site Competition in Compound Mixtures.", JAM. CHEM. SOC., vol. 126, 2004, pages 15495 - 15503, XP002461719, DOI: doi:10.1021/ja048365x
ARMSTRONG ET AL., CANCER CELL, vol. 3, 2003, pages 173 - 83
BELOKON ET AL., TETRAHEDRON ASYMM., vol. 9, 1998, pages 4249 - 4252
BRUNEI ET AL., CHEM. COMMUN., 2005, pages 2552 - 2554
BRUNEL ET AL., CHEM. COMMUN., 2005, pages 2552 - 2554
CHITTENDEN ET AL., EMBO, vol. 14, 1995, pages 5589
DEITERS ET AL., J. AM. CHEM. SOC., vol. 125, 2003, pages 11782 - 11783
DEITERS ET AL., JAM. CHEM. SOC., vol. 125, 2003, pages 11782 - 11783
FIELDS ET AL.: "Synthetic Peptides: A User's Guide", 1992, W. H. FREEMAN & CO., pages: 77
FIESER; FIESER: "Fieser and Fieser's Reagents for Organic Synthesis", 1994, JOHN WILEY AND SONS
GALANDE ET AL., J. COMB. CHEM., vol. 7, 2005, pages 174 - 177
GREENAWAY J. ET AL., MOL. CANCER THER., vol. 8, 2009, pages 64 - 74
GREENE; WUTS: "Protective Groups in Organic Synthesis, 2d. Ed. ,", 1991, JOHN WILEY AND SONS
GRUBBS ET AL.: "Ring Closing Metathesis and Related Processes in Organic Synthesis", ACC. CHEM. RES., vol. 28, 1995, pages 446 - 452, XP002035670
GUAN, J. ET AL., CANCER CHEMO PHARMA, 24 December 2008 (2008-12-24)
HUNT: "Chemistry and Biochemistry of the Amino Acids", 1985, CHAPMAN AND HALL, article "The Non-Protein Amino Acids"
JENKINS, D. E. ET AL., CLIN. & EXP. METASTASIS, vol. 20, 2003, pages 745 - 756
JOURNAL OF THE NATIONAL CANCER INSTITUTE, vol. 97, no. 5, 2 March 2005 (2005-03-02), pages 330
LAROCK: "Comprehensive Organic Transformations", 1989, VCH PUBLISHERS
LELEKAKIS, M. ET AL., CLIN & EXP METASTASIS, 1999, pages 163 - 170
MANNHOLD R, KUBINYI H, FOLKERS G: "Methods and Principles in Medicinal Chemistry", WILEY
MOSBERG ET AL., J. AM. CHEM. SOC., vol. 107, 1985, pages 2986 - 2987
MOSBERG ET AL., J. AM.CHEM. SOC., vol. 107, 1985, pages 2986 - 2987
MUSTAPA, M.; FIROUZ MOHD ET AL., J. ORG. CHEM, vol. 68, 2003, pages 8193 - 8198
OR ET AL., J. ORG. CHEM., vol. 56, 1991, pages 3146 - 3149
PAQUETTE,: "Encyclopedia of Reagents for Organic Synthesis", 1995, JOHN WILEY AND SONS
PUNNA ET AL., ANGEW. CHEM. INT. ED., vol. 44, 2005, pages 2215 - 2220
RASMUSSEN ET AL., ORG. LETT., vol. 9, 2007, pages 5337 - 5339
ROSTOVTSEV ET AL., ANGEW. CHEM. INT. ED., vol. 41, 2002, pages 2596 - 2599
SCATENA C. D. ET AL., PROSTATE, vol. 59, 2004, pages 292 - 303
SCHAFMEISTER ET AL., J. AM. CHEM SOC., vol. 122, 2000, pages 5891
SCHAFMEISTER ET AL., J. AM. CHEM. SOC., vol. 122, 2000, pages 5891 - 5892
SCHAFMEISTER; VERDINE, J. AM. CHEM. SOC., vol. 122, 2005, pages 5891
SEEBACH ET AL., ANGEW. CHEM. INT. ED. ENGL., vol. 35, 1996, pages 2708 - 2748
SZEWCZUK ET AL., INT. J. PEPTIDE PROTEIN RES., vol. 40, 1992, pages 233 - 242
THALLINGER ET AL., CLIN. CANCER RES., vol. 10, 2004, pages 4185 - 4191
TOMOE ET AL., J. ORG. CHEM., vol. 67, 2002, pages 3057 - 3064
TORNOE ET AL., J. ORG. CHEM., vol. 67, 2002, pages 3057 - 3064
VAICKUS, CRIT REV. ONCOL./HEMOTOL., vol. 11, 1991, pages 267 - 97
WALENSKY ET AL., MOL CELL, vol. 24, 2006, pages 199 - 210
WALENSKY ET AL., SCIENCE, vol. 305, 2004, pages 1466
WALENSKY ET AL., SCIENCE, vol. 305, 2004, pages 1466 - 1470
WALENSKY ET AL., SCIENCE, vol. 305, 2004, pages 1466 - 70
WANG ET AL., GENES DEV., vol. 10, 1996, pages 2859
WANNER K, HÖFNER G:: "Mass Spectrometry in Medicinal Chemistry", 2007, WILEY-VCH, article D. A. ANNIS; C.-C. CHUANG; N. NAZEF: "ALIS: An Affinity Selection-Mass Spectrometry System for the Discovery and Characterization ofProtein-Ligand Interactions", pages: 121 - 184
WILLIAMS ET AL., J. AM. CHEM. SOC., vol. 113, 1991, pages 9276
YANG ET AL., METHODS ENZYMOL., vol. 130, 1986, pages 208
YANG, BIN ET AL., BIOORG MED. CHEM. LETT., vol. 14, 2004, pages 1403 - 1406
ZHANG ET AL., J. AM. CHEM. SOC., vol. 127, 2005, pages 15998 - 15999

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GB201014734D0 (en) 2010-10-20
CA2714251A1 (fr) 2009-08-13
US20190071469A1 (en) 2019-03-07
CN101980718A (zh) 2011-02-23
BRPI0907754A2 (pt) 2015-07-21
US8808694B2 (en) 2014-08-19
IL207421A0 (en) 2010-12-30
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CA2714251C (fr) 2017-08-15
US20150038430A1 (en) 2015-02-05
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